Light Robotics Structure-mediated Nanobiophotonics

Jesper Glückstad

Darwin Palima

Series Editor

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xv

List of Contributors

Badri L. AekboteBiological Research Centre, Szeged, Hungary

Fumihito AraiNagoya University, Nagoya, Japan

Andrew Rafael BañasTechnical University of Denmark, Lyngby, Denmark

Azra BahadoriNiels Bohr Institute, University of Copenhagen, Copenhagen, Denmark

Álvaro Barroso PeñaInstitute of Applied Physics, University of Muenster, Muenster, Germany

Poul M. BendixNiels Bohr Institute, University of Copenhagen, Copenhagen, Denmark

Konrad BerghoffUniversity of Bayreuth, Bayreuth, Germany

Ann A.M. BuiThe University of Queensland, St Lucia, Brisbane, QLD, Australia

András BuzásBiological Research Centre, Szeged, Hungary

David CarberryThe University of Queensland, St Lucia, Brisbane, QLD, Australia

Duncan CaseyCentre for Functional Nanomaterials, University of Bristol, United Kingdom

Cornelia DenzInstitute of Applied Physics, University of Muenster, Muenster, Germany

Cemal EsenApplied Laser Technologies, Ruhr-Universität Bochum, Universitätsstraße, Bochum, Germany

Donglei (Emma) FanMaterials Science and Engineering Program; The University of Texas at Austin, Austin, TX, United States

Lisa GebhardtUniversity of Bayreuth, Bayreuth, Germany

Jesper GlückstadTechnical University of Denmark, Lyngby, Denmark

xvi List of Contributors

Rachel GrangeOptical Nanomaterial Group, Institute for Quantum Electronics, Zurich, Switzerland

Wolfgang GrossUniversity of Bayreuth, Bayreuth, Germany

Jianhe GuoMaterials Science and Engineering Program, The University of Texas at Austin, Austin, TX, United States

Simon HannaUniversity of Bristol, Bristol, United Kingdom

Takeshi HayakawaNagoya University, Nagoya, Japan

Antoine HouillotThe University of Queensland, St Lucia, Brisbane, QLD, Australia

Jannis KöhlerApplied Laser Technologies, Ruhr-Universität Bochum, Universitätsstraße, Bochum, Germany

Neil M. KadUniversity of Kent, Canterbury, Kent, United Kingdom

Anatolii V. KashchukThe University of Queensland, St Lucia, Brisbane, QLD, Australia

Lóránd KelemenBiological Research Centre, Szeged, Hungary

Steve KellerUniversity of Bayreuth, Bayreuth, Germany

Holger KressUniversity of Bayreuth, Bayreuth, Germany

Sarah I. KsouriApplied Laser Technologies, Ruhr-Universität Bochum, Universitätsstraße, Bochum, Germany

Hisataka MaruyamaNagoya University, Nagoya, Japan

Mark NeilImperial College London, London, United Kingdom

Timo A. NieminenThe University of Queensland, St Lucia, Brisbane, QLD, Australia

Lene B. OddershedeNiels Bohr Institute, University of Copenhagen, Copenhagen, Denmark

xviiList of Contributors

Pál OrmosBiological Research Centre, Szeged, Hungary

Andreas OstendorfApplied Laser Technologies, Ruhr-Universität Bochum, Universitätsstraße, Bochum, Germany

Darwin PalimaTechnical University of Denmark, Lyngby, Denmark

David PhillipsUniversity of Glasgow, United Kingdom

Mark R. PollardDFM A/S, K. Lyngby, Denmark

Daryl PreeceUniversity of California San Diego, La Jolla, CA, United States

Halina Rubinsztein-DunlopThe University of Queensland, St Lucia, Brisbane, QLD, Australia

Anton SergeyevOptical Nanomaterial Group, Institute for Quantum Electronics, Zurich, Switzerland

Linda ShiUniversity of California San Diego, La Jolla, CA, United States

Stephen SimpsonInstitute of Scientific Instruments of the CAS, Brno, Czech Republic

Alexander B. StilgoeThe University of Queensland, St Lucia, Brisbane, QLD, Australia

Mark Jayson VillangcaTechnical University of Denmark, Lyngby, Denmark

Gaszton VizsnyiczaiBiological Research Centre, Szeged, Hungary

Douglas WylieImperial College London, London, United Kingdom

Shu ZhangThe University of Queensland, St Lucia, Brisbane, QLD, Australia

Gordon ZylaApplied Laser Technologies, Ruhr-Universität Bochum, Universitätsstraße, Bochum, Germany

xix

Biographies

Jesper Glückstad is Professor and Group Leader at the Department of Photonics Engineering at the Technical University of Denmark. He established the Program-mable Phase Optics Laboratory in Denmark in the late 1990s, and served as Guest Professor in Biophotonics at Lund Institute of Technology, Sweden, 2006–11. In 2004 he received the prestigious Doctor of Science (DSc) degree from the Techni-cal University of Denmark. Prior to his achievements in Denmark, he was a vis-iting scientist at Hamamatsu Photonics Central Research Laboratories and in the Physics Department at Osaka University in Japan. He is the year 2000 recipient of the Danish Optical Society Award and was elected as Scientist of the Year in 2005 by Dir. Ib Henriksen’s Foundation in Denmark. Prof. Glückstad is a 2010 elected Fellow of the OSA and a Fellow of the SPIE as the first from Denmark. Between 2012 and 2014, he served on the prestigious SPIE Fellows committee. Since he ob-tained his PhD at the Niels Bohr Institute in 1994, he has published more than 300 journal articles and international conference papers among them several in Nature journals. He holds more than 30 international patent inventions to his name and is founder of the 2011 spin-out OptoRobotix ApS and its recent associated tech-transfer unit GPC Photonics.

Darwin Palima is Associate Professor in the Department of Photonics Engineering at the Technical University of Denmark where he teaches a course he created on Biophotonics and Optical engineering. He has pioneered new scientific directions at the programmable phase optics laboratory and, as lab responsible, closely men-tors the group’s PhD students and postdocs. He collaborates extensively with Jesper Glückstad from joint supervision of PhD students to joint authorships of scientific papers, conference presentations, patent applications, and popular articles. Having authored a physics textbook before moving to Denmark, he played a key role in their jointly authored monograph on Generalized Phase Contrast. The present volume is another product of their collaboration. Darwin concurrently taught at the Philippine Science High School when he worked through his PhD at the University of the Philippines proposing improvements to computer-generated holography. His interest in structured light started in the mid-1990s when he studied optical vortices for his BSc thesis.

xxi

Preface

Scientific disciplines constantly evolve and create new offspring—subdisciplines—that combine the favorable characteristics from its forerunners. The merger of biology and photonics has within the last decade produced one such offspring, Biophotonics, which harnesses light to study biological materials. More recently we have seen the exciting merger of biophotonics with contemporary nanophotonics into so-called nanobiophotonics culminating with the 2014 Chemistry Nobel Prize for superresolution microscopy—now simply coined nanoscopy. The usage of the term Biophotonics in scientific papers curiously began to shoot up after Prasad’s 2003 book “Introduction to Biophotonics,” the first monograph on the field. The aforementioned correlation can mean two things: (1) The book was written at an opportune time when biophotonics was actually taking off; and (2) the book helped clarify the scope of the emerging field for disparate researchers who then contributed to its growth upon realizing how their activities are united in a new context.

This book was written along similar lines on the borderline between a plurality of emerging scientific subdisciplines. After years of working on light-driven trapping and manipulation, we can see that a confluence of developments is now ripe for the emer-gence of a new area that can contribute to nanobiophotonics—Light Robotics—which combines advances in microfabrication and optical micromanipulation together with intelligent control ideas from robotics. This volume collects expert contributions from various areas that are coherently coming together through light robotics. We cover the fundamental aspects of optical trapping systems, microfabrication, and microassem-bly, and present theoretical principles and experimental illustrations for optimizing the optical force and torque. We also present an array of various new functionalities that are enabled by these new designed structures—light-driven microrobots. Finally, we cover various nanobiophotonics applications demonstrating the use of biophysical tools utilizing light robotics concepts.

We have endeavored to make the book accessible to a broad audience from ad-vanced undergraduates and graduate students to practitioners and researchers not only in nanobiophotonics and micro- and nanotechnology, but also to other areas in optics and photonics, mechanical engineering, control and instrumentation engineer-ing, and related fields. We hope that this book is able to do justice in presenting a clearer picture of this emerging field, which is essential to igniting the needed syn-ergy between various stakeholders in the development of this field. We are fortunate to be taking these first steps together with all the contributors from across four conti-nents to whom we owe a debt of gratitude for the time, effort, and expertise devoted into formulating their respective chapters. Thank you very much!

Jesper GlückstadDarwin Palima

Technical University of Denmark, Kgs. Lyngby, DenmarkMay 2017

xxiii

Introduction

Darwin Palima, Jesper GlückstadTechnical University of Denmark, Kgs. Lyngby, Denmark

The 1920 science fiction play, R.U.R, introduced the word robot to describe mass-produced synthetic humans [1]. The fascination with intelligent humanoid robots continues today and remains a favorite theme in science fiction. Beyond fiction, the modern definition of a robot, according to ISO 8373:2012 [2], is an “actuated mecha-nism programmable in two or more axes with a degree of autonomy, moving within its environment, to perform intended tasks” and robotics is the “science and practice of designing, manufacturing, and applying robots.” A robotic device is similar to a robot but may lack in the number of programmable axes or autonomy, that is, teleop-erated devices controlled by human operators as we see in robotic surgery [3]. This book shows that various light-based technologies are now enabling functionalities in what can be aptly recognized as an emerging field of light robotics. In the new con-text of light robotics, it will suffice to use robots and robotic devices interchangeably.

Light robotics refers to the use of light to realize functionalities associated with robotics and robotic devices or, alternately, importing ideas from robotics to develop new light-based functionalities. This book focuses on functionalities that are relevant to biophotonics at the micro- and nanoscale. However, just as what we call optical tweezers does not look remotely close to mechanical tweezers, one can expect that many of the microrobots that we will encounter in this book do not resemble conven-tional robots or robotic devices. Nonetheless, a closer look will reveal that they have a role to play in realizing light-based robotics for biophotonics.

A robot needs a control system to monitor and regulate its functions and provide an interface to users and other equipment, if necessary. However, working in the mi-cro- and nanoscale biophotonics regime imposes restrictive conditions on how much hardware and software we can fit into the tiny microstructures—the microrobots. We can work around this basic space constraint by off-loading the intensive computing and control aspects to regular computers. This off-loading is analogous to how com-pact, highly portable devices gain access to sophisticated computational power via cloud computing services. We can further optimize the limited space by also removing the onboard power.

So, how can we actuate microrobots having no onboard processor and power source? Light is a useful agent for remote actuation. Using light as information carrier would have worked to transmit coded instructions to an onboard processor on a powered robot [4]. Without onboard power, we could use light as energy carrier to supply power remotely via onboard photovoltaics [5]. Without onboard circuitry, we can still use light to carry energy to directly drive light-sensitive structures using materials that can convert optical to mechanical energy via photomechanics [6,7]. Taking off from optical trapping and optical micromanipulation, this book focuses on approaches that exploit light’s momentum to drive microscopic objects designed

xxiv Introduction

as microrobots [8]. Most modern optical micromanipulation derive inspiration from Arthur Ashkin’s pioneering work in 1970, which presented the possibility of using optical radiation pressure to trap and manipulate microparticles [9]. One popular implementation, the optical tweezers, uses a sharply focused beam to work as actua-tor to trap and manipulate microparticles in 3D [10,11]. However, there are many possible implementation geometries, each having their respective strengths and weaknesses [12]. Designing bespoke light robotics systems must consider the appli-cation-specific requirements and constraints.

Fig. 1 presents a schematic showing the different elements in a light robotics system. One of the key elements is controlled light delivery to actuate and adjust the position of a microrobot. The microrobot is shown carrying a functional load, analogous to an end effector—the device installed at the end of robotic arms that interacts with the environment to perform its task. The microrobot and its functional load may themselves be fabricated or assembled using controlled light delivery. With clever design, the microrobot and its functional load can enable structure-mediated access either to nanoscopic length scales beyond the reach of diffraction-limited light beams or to minimize direct exposure when probing light-sensitive processes. Con-trolled light delivery may also be used to directly interact with the system as well as to activate and regulate the functional load, which can be designed either to deliver stimuli or collect information, that is, for sensing, detection, imaging, or spectros-copy in conjunction with an auxiliary system. In general, there can be a swarm of microrobots simultaneously working at different tasks. These tasks can be geared to-ward biophotonics, a field that aims to harness light to image, detect, and manipulate biological matter for fundamental studies and various applications [13]. This book explores the various aspects of the light robotics system and presents illustrative examples harnessing their utility for biophotonics.

FIGURE 1 Schematic of a Light Robotics System

(inset artist’s rendition courtesy of A. Bañas).

xxvIntroduction

Optical tweezers are precision biophysical tools that can measure the mechanical properties of biomolecules. This is typically done by tethering them onto micro-spheres held by optical tweezers exerting calibrated optical forces. This illustrates a light robotics approach in biophotonics. This use of optically trapped microspheres is a well-developed subject in optical trapping and is treated at length in several books on optical tweezers [14,15]. In this book, our focus is on leveraging advances in microfabrication to design microrobots that elevate the level of control and realize new functionalities. We will still discuss trapped microspheres but the focus is on alternate functionalities that they can offer beyond calibrated optical forces.

Telerobotics has proven indispensable for distances that are orders of magnitude larger than what we can conveniently access, as the success of the mars exploration rovers shows [4]. However, working at orders of magnitude smaller length scales arguably presents a similar challenging inaccessibility and remoteness. Thus, nano-manipulation techniques are also referred to as telenanorobotics approaches, for instance, when using atomic force microscopy (AFM) as the nanorobot [16–18]. Besides autonomous nanoscopic machines, an alternate definition of nanorobots and nanorobotics includes complete systems, regardless of actual size, that can manipu-late nanoscopic entities with nanoscale precision. We adopt this duality in this book and use microrobots and microrobotics when referring both to the tiny light-driven microstructures as well as the complete systems used to optically control and ma-nipulate them.

THE BOOK IN BRIEFBack in 2005, Elliot Botvinick and Michael Berns demonstrated RoboLase, a teleop-erated system at the University of California, where internet-based users as far away as Australia could control the RoboLase microscope and its laser beams for abla-tion and optical micromanipulation [19]. In Chapter 1, project scientists at Michael Berns’ lab discuss the basic concepts and design considerations in optical trapping and manipulation systems, including the RoboLase. The chapter provides special focus on the different human interfaces that are being explored to help operators achieve intuitive control in light robotics.

Versatile technologies for microfabrication and microassembly are essential for rapidly prototyping new design ideas in light robotics. In this regard, Chapter 2 discusses the fundamentals of two-photon fabrication, a 3D laser direct writing tech-nique, as well as various light-based assembling techniques that exploit the pick-and-place robotic functionality of optical tweezers for bottom-up fabrication using basic components. This chapter outlines different methods for fixed assembly and illustrates examples of detachable assembly for reconfigurability in lab-on-chip ap-plications. The authors use holographic optical tweezers to simultaneously generate multiple individually controllable optical tweezers for experimental demonstrations.

Chapter 3 details the theory on the behavior of complex shapes in optical fields and discusses their rigorous force calibration. It introduces the concept of

xxvi Introduction

shape-induced forces, which treats the shape of the microobject as a design param-eter that can be optimized for applications, for example, to maintain the same force even when the object moves relative to the illuminating beam. The chapter describes the fabrication, 3D tracking and optical control of these microtools together with a range of demonstration experiments including 3D surface imaging for applications in nanobiophotonics.

Designing nonspherical structures enables control not only over the optical force but the optical torque as well. Chapter 4 reviews the basic theory/principles for rotat-ing microstructures with light and presents heuristic principles for designing light-driven micromachines. Asymmetry is a key element: A spherically symmetric object cannot be rotated by optical illumination. The chapter illustrates these concepts for a prototypical optically driven micromachine, which it follows through fabrication and experimental demonstration. To appreciate these developments in a larger con-text, Chapter 5 reviews the recent progress in the development of electromagnetic wave enabled micro/nanomechanical motors. To provide expanded benchmarking, the chapter also considers recent developments in plasmonics-enabled nanorotors as well as optoelectronic and electric robotics micro- and nanodevices.

The combination of advances in microfabrication with optical manipulation creates various novel functionalities for light robotics and we see some of them in several of the chapters that follow. Chapter 6 presents two-photon fabricated struc-tures that are functionalized with specific physical, chemical, and biochemical meth-ods to create complex microtools. Light-driven structures are used in synthetic model systems for understanding the dynamics of self-propelled microswimmers and hy-drodynamic synchronization. Moreover, fabrication incorporates various functional elements into the light-actuated microrobot, such as waveguides, SERS probes and even contact-based grippers for handling cells with minimal illumination.

Chapter 7 demonstrates various light driven micro- and nanorobots for cellular transportation, force application, cell surgery, and extra- and intracellular measure-ments. Structures doped with temperature-sensitive dyes can act as local temperature sensors. Nanowires exhibiting high photothermal effect work as efficient and highly localized light-activated heaters that are steered by microrobots, for example, to po-rate cell membranes. The chapter discusses the fabrication of microrobots carrying nanowires as their functional load, which opens a gateway for bringing advances in nanowires into light robotics. To present a glimpse of opportunities available in nanowire photonics [20], Chapter 8 discusses the optimization of lithium niobate nanowires for second harmonic generation and waveguiding and experimentally demonstrates that the guided second harmonic light can be used for localized fluo-rescence excitation. Finally, Chapter 9 looks at the various opportunities available from metallic nanolayers that light robotics can potentially tap from. For illustration, the chapter considers a hollow microrobot with a metallic disc nanolayer deposited inside. Experimental demonstrations illustrate that this effectively works as a light-activated syringe that a microrobot can use to load and unload cargo. This can be especially useful for cargo that may be difficult to trap, for example, due to size or poor refractive index contrast.

xxviiIntroduction

The final section of the book illustrates the strong potential of light robotics in biophotonics through various example applications demonstrating the use of light robotics concepts for developing biophysical tools to the investigation of various aspects of biophotonics.

Chapter 10 sets the stage and examines an important area where light robotics can play a strong role: investigating biological processes with resolution in the single-cell and subcellular levels to reveal details that are lost in standard protocols employ-ing a large ensemble of cells. The chapter surveys various tools and techniques that interact with the cell membrane, disrupting it either via direct laser illumination or structure-mediated approach. Different opportunities are illustrated, such as opti-cal poration and lysis alongside surface chemistry and coatings capable of causing structural effects within a target cell’s plasma membrane. Chapter 11 explores in detail the use of gold nanoparticles as functional elements that can be positioned and heated with light to perturb cellular membranes. Plasmonic effects turn the gold nanoparticles into efficient nanoheaters that can weld cells in an altogether different manner: instead of bonding cells together, it triggers the fusion of the cell membranes to combine intracellular materials into a single viable hybrid cell. This hybridization offers new opportunities in basic and applied cellular research. The technique also works for introducing foreign materials into cells by fusing them with synthesized vesicles. Going further down the size scale, Chapter 12 uses light robotics in a single-molecule application. This chapter takes readers through the various development aspects from fabrication to the calibration and final application of optically trapped probes in single-molecule biology force measurements using DNA filaments and a DNA repair protein.

Chapter 13 explores biophysical tools created using two types of functional loads: heaters and chemical sources. Here, light robotics is used to control, both temporally and spatially, the temperature and chemical gradients established in a cel-lular environment. The chapter also develops various optical and numerical analysis tools to quantitatively characterize the cellular response to the thermal and chemical gradients, respectively called thermotaxis and chemotaxis. The promising results in this chapter indicate that light robotics can play a role in understanding the basic processes governing cellular migration, which can have rich implications from controlling biofilm formation to regulating immune system response. However, this understanding can also have a symbiotic benefit to the light robotics field, particu-larly in developing autonomous microrobots. Chapter 14 discusses work along this direction. As we saw in Fig. 1, controlled light delivery can be used to fabricate/assemble microrobots that, in turn, may also be steered and functionalized by light. However, light scattering makes controlled light delivery increasingly difficult as we go deeper into biological matter. Thus, working inside thick biological tissues will require either a light delivery system beyond the current state of the art or au-tonomous micrororobots that are capable of autonomous navigation. Given the space constraints discussed earlier, we can turn to biomimetics for solutions that mimic processes governing cellular migration through various field gradients. Chapter 14 uses controlled light delivery to assemble a hybrid microrobot that consists of a

xxviii Introduction

designed structure (e.g., a drug carrier) and several bacteria preferentially arranged to propel the microrobot using their flagellar motion. Moving forward requires devel-oping microrobots that can efficiently respond and move according to task-specific gradients.

OUTLOOK: CHALLENGES AND OPPORTUNITIESThe various elements of light robotics outlined in Fig. 1 present various challenges and opportunities. Advancing light robotics can benefit from improvements in optical manipulation–for example, designed structures that are highly efficient in translat-ing incident light into usable optical force/torque and new strategies for controlled delivery that can work reliably through highly scattering biological media. However, optical manipulation is just one aspect of light robotics. Further progress will re-quire support from proactive research and development of compatible light sources, sensors, and other allied technologies to equip the microrobots with needed function-alities. Optical microfabrication and microassembly can be enriched by ideas from other microfabrication and assembly techniques to develop the next generation of microrobots. However, real progress requires not just isolated advances in these sub-disciplines but also dedicated efforts at system integration. Moreover, applications in biophotonics will require a strong collaboration between light robotics tool develop-ers and biological end-users. Newly developed materials, tools, and techniques need to be subjected to rigorous biocompatibility and cell viability studies. Collaboration would also be essential in guiding the creation of light robotics tools that genuinely address authentic users’ needs. Bringing these tools out of the optics and photonics labs and into biophotonics end-users, will require instruments that can be operated intuitively by the end-users and so attention to ergonomics and intuitive human in-terfaces is needed as well.

This book aims to promote this collaboration. We have collected contributions from various traditional research fields and presented how they can coherently come together through light robotics. We hope this book can contribute to igniting a similar synergy between the different stakeholders, which will be needed to accelerate devel-opments in this new field. There are also opportunities for making headway through light robotics in other application areas beyond biophotonics.

REFERENCES[1] Capek K, R.U.R. (Rossum’s Universal Robots), Prague, CZ, 1920.[2] ISO 8373:2012—Robots and robotic devices—Vocabulary. Available from: http://www.

iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnumber=55890[3] Lanfranco AR, Castellanos AE, Desai JP, Meyers WC. Robotic surgery: a current

perspective. Ann Surg 2004;239:14–21. [4] Mars exploration rover mission: home. Available from: http://mars.nasa.gov/mer/home/

xxixIntroduction

[5] Jones BE, McKenzie JS. A review of optical actuators and the impact of micromachin-ing. Sensors Actuators A Phys 1993;37:202–7.

[6] Yu Y, Nakano M, Ikeda T. Photomechanics: directed bending of a polymer film by light. Nature 2003;425:145.

[7] Han DD, Zhang YL, Ma JN, Liu YQ, Han B, Sun HB. Light-mediated manufacture and manipulation of actuators. Adv Mater 2016;28:8328–43.

[8] Palima D, Glückstad J. Gearing up for optical microrobotics: micromanipulation and ac-tuation of synthetic microstructures by optical forces. Laser Photon Rev 2013;7:478–94.

[9] Ashkin A. Acceleration and trapping of particles by radiation pressure. Phys Rev Lett 1970;24:156–9.

[10] Ashkin A, Dziedzic JM, Bjorkholm JE, Chu S. Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett 1986;11:288.

[11] Ashkin A, Dziedzic JM. Optical trapping and manipulation of viruses and bacteria. Science 1987;235:1517–20.

[12] Glückstad J, Palima D. Generalized phase contrast: applications in optics and photonics. In: Springer Series in optical sciences, vol 146, Netherlands:Springer; 2009.

[13] Shen X, Van Wijk R, editors. Biophotonics: optical science and engineering for the 21st century. New York: Springer; 2005.

[14] In: Padgett M, Molloy J, McGloin D, editors. Optical Tweezers: Methods and Applica-tions, vol. 20105644. Boca Raton: Chapman and Hall/CRC; 2010.

[15] Jones PH, Marago OM, Volpe G. Optical tweezers: principles and applications. Cambridge: Cambridge University Press; 2015.

[16] Sitti M, Hashimoto H. Tele-nanorobotics using an atomic force microscope as a nanorobot and sensor. Adv Robot 1998;13:417–36.

[17] Sitti M, Hashimoto H. Tele-nanorobotics using atomic force microscope. In: Proceedings of 1998 IEEE/RSJ international conference on intelligent robots and systems. Innovations in theory, practice and applications (Cat. No. 98CH36190). IEEE 1998; 3. pp. 1739–46.

[18] Xie H, Onal C, Régnier S, Sitti M. Atomic force microscopy based nanorobotics. In: Springer tracts in advanced robotics, vol. 71. Berlin, Heidelberg: Springer; 2012.

[19] Botvinick EL, Berns MW. Internet-based robotic laser scissors and tweezers microscopy. Microsc Res Tech 2005;68:65–74.

[20] Yan R, Gargas D, Yang P. Nanowire photonics. Nat. Photonics 2009;3:569–76.

3

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00001-XCopyright © 2017 Elsevier Ltd. All rights reserved.

Daryl Preece, Linda ShiUniversity of California San Diego, La Jolla, CA, United States

CHAPTER OUTLINE

1 Optical Tweezers Basics .........................................................................................41.1 Optical Tweezers ................................................................................41.2 Optical Gradient Force .......................................................................41.3 Practical Setup ..................................................................................51.4 Forces ..............................................................................................6

2 Measurement of Position and Force ........................................................................62.1 Drag Force Method .............................................................................72.2 Equipartition .....................................................................................82.3 Langevin Method ...............................................................................82.4 Light Deflection Method .....................................................................9

3 System Design and Instrumentation of Optical Manipulation Systems ..................... 103.1 System Design .................................................................................103.2 System Implementation ....................................................................12

4 Human Interfaces ................................................................................................ 154.1 Software Control of Optical Manipulation Systems ...............................15

5 Control with Peripheral Devices ........................................................................... 166 3D Control .......................................................................................................... 19

6.1 Gathering Spatial Information ...........................................................206.2 Supplying 3D Information .................................................................20

7 Haptics ............................................................................................................... 228 Internet Control—Controlling Systems Remotely ................................................... 249 Future Directions ................................................................................................. 26References ................................................................................................................ 27

Human gesture recognition for optical manipulation and its future nanobiophotonics applications

1

4 CHAPTER 1 Human gesture recognition for optical manipulation

1 OPTICAL TWEEZERS BASICS1.1 OPTICAL TWEEZERSWith the invention of the laser, it became practical to focus high intensities of light down to a spot size of a few hundred nanometers. At this scale, optical forces become significant enough to move small particles. Arthur Ashkin did the first work in this area while working at Bell Labs in 1970. Ashkin used a 514 nm (CW) argon ion laser to push transparent latex spheres suspended in water and a counter-propagating geometry to produce a stable optical trap [1]. In 1971, Ashkin published a further paper showing that particles could also be levitated by what he called an optical fountain [2]. However, Ashkin noticed that particles were also pulled into the center of the beam. The force behind this phenomenon, the “optical gradient force,” seemed to pull small dielectric particles from 10 µm down to 25 nm into the brightest part of the beam. In 1986, Ashkin exploited this force to create the single-beam gradient trap [3]. The trap gave researchers control over particles that were previously too small to manipulate by other means allowing them to exert piconewton forces over nanometer length scales.

1.2 OPTICAL GRADIENT FORCETo understand the single-beam gradient trap in an intuitive way, one must imagine a particle in close proximity to a highly focused beam of light (Fig. 1.1). We con-sider that the refractive index of the particle is higher than the refractive index of the surrounding medium. When light hits the particle, some proportion of the light will pass through the spherical surface of the particle causing it to act as a lens. If the particle is displaced sideways from the center of the beam, transmitted light will be directed in the same sense as the displacement. The resultant force on the particle due to this deflection is backward toward the center of the beam (Fig. 1.1B and C). In other words in the direction of the positive gradient of intensity and it is thus known as the “gradient force.” The rest of the light, which is reflected or has momentum contributions, which are not toward the field gradient, will contribute to a scattering force. The scattering force is proportional to the optical intensity and points in the direction of the incident light [4]. Since the gradient force is greater than scattering force, the net result is a force pulling the particle along the gradient of intensity and toward the highest intensity.

If the trapping beam is highly focused, then the particle will also experience a force along the axis of the beam propagation. Since the convergence or divergence of the exiting rays change with the particle’s distance from the focal point so do the axial momentum components (Fig. 1.1A and D). This means that for a particle below the focus of the beam, the forces resolve to push the particle back toward the focus of the beam and vice versa for particles above the beam focus. The particle can thus be trapped in three dimensions.

51 Optical tweezers basics

1.3 PRACTICAL SETUPIt is fairly easy to construct a simple single beam gradient force trap. In order to cre-ate a highly focused beam, a microscope objective is usually used. This can be used both for trapping and viewing of the sample. The objective lens should be of high numerical aperture (NA) to ensure that the gradient force outweighs both gravity and the scattering force. It should be noted that many tweezers systems tend to be inverted in order that the scattering force acts against gravity allowing lower laser powers to be used. For increased NA, water or oil immersion objectives are often used [5]. The practical NA of the beam is usually fully exploited by overfilling the back aperture of the objective lens, though this must be weighed against power con-siderations [6] (Fig. 1.2).

The sample may be held on a translation stage, which may be moved to enable viewing of different parts of the sample and can be used to translate the optical trap to areas of interest. Standard Köhler illumination may be used to illuminate the sample. Though other modalities of optical trap exist, such as fiber traps [7], the single-beam gradient trap represents the most common type of optical trap. Often the single beam trap is extended to counter propagating optical traps, which can provide a greater optical force at a lower NA [8,9].

FIGURE 1.1 Diagram Showing a Particle at Different Places in Focused Gaussian Laser Beam

Light exits the particle with a different momentum than it enters with causing the particle to move. (A), (B), (C), and (D) show a particle at different points in the beam with ray tracing of the refracted light. As rays are redirected by the particle, the momentum components of the rays change, the resultant force on the particle is shown by the arrows at the center of each particle..

6 CHAPTER 1 Human gesture recognition for optical manipulation

1.4 FORCESA number of different ways have been developed to parameterize optical forces. Ashkin developed the simple metric of the force efficiency “Q” to do this [4]. The time-averaged force acting upon a particle can be expressed as:

=F QnP

c

where n is the refractive index of the surrounding medium, P is the beam power at the focus, c is the speed of light in free space, and Q is the dimensionless force efficiency. The force efficiency can also be viewed as the fractional momentum change per photon.

For smaller particles, Rayleigh or dipole scattering models may provide a better way of predicting optical forces. Though designed with a point dipole in mind, these models can produce good results for particles less than a wavelength of light in size [10]. For bigger particles, Mie scattering theory can be used to predict optical forces [11]. For particles of sufficient size, ray tracing may be applicable [12–14]. When dealing with more complex problems, such as nonspherical or complex particles, a T-matrix method may be used [15,16]. For complicated shapes, the Maxwell stress tensor method may also be useful [17].

2 MEASUREMENT OF POSITION AND FORCEPrecise control of light-driven systems cannot be accomplished without an accurate understanding of forces present in the system. This is particularly challenging when dealing with objects that may move only a matter of nanometers. In optical tweezers,

F=QnPc

FIGURE 1.2 A Basic Optical Tweezers System With a 1064 nm Laser Source, Beam Steering Mirror, 4f Imaging System, Dichroic Mirror, CCD Camera, High NA Objective Lens, Illumination Light, and a Condenser Lens

72 Measurement of position and force

displacements may be sensed to nanometer accuracy and forces have been calibrated down to 25 fN [18], making optical traps extremely sensitive sensors.

Empirically, it has been shown [19] that an optically trapped particle behaves like a damped mass in a parabolic energy well where displacements from the trap center are small. This behavior is analogous to a mass attached to a Hookean spring. However, Simmons and coworkers found (for 1 µ m beads) that for displacements that are greater than half a bead radii, the restoring force rapidly becomes nonlinear reaching a peak at roughly the bead radius. This is consistent with calculations done by Ashkin [4] but this distance may expand as the bead diameter shrinks into the Rayleigh regime (a<<λ).

κ= −F xopt

The force constant k is proportional to the trap stiffness which depends on a number of factors, such as the trapping geometry; particle size and refractive index; refractive index of the media; and the overall laser power. If the force constant can be calibrated, it is possible to measure the absolute optical force exerted on an object.

A variety of techniques have been developed for ascertaining the forces exerted by optical traps. We will now discuss several commonly used methodologies, which have been widely employed. In choosing an appropriate method, it is important to consider external noise sources, such as vibrations, measurement time scales, and equipment constraints, such as stage frequency response or data acquisition band-width. Generally, all methods can be used in most trapping systems but some may require specific equipment in order to get precise measurements. The reader is en-couraged to explore the references for more information.

2.1 DRAG FORCE METHODIn most cases, the optical force is not the only force acting on the particle. Thermal forces bombard the particle causing it to move by Brownian motion. This motion is impeded by the viscous drag of the particle. For a particle in an over damped media where there is no turbulence (i.e., low Reynolds number, Re < 1), the drag force can be represented by the Stokes drag force that linearly scales with velocity.

γ γ π η= =F v awhere 6drag 0 0

Where a is the radius of the particle, η is the dynamic viscosity of the media, and v is the velocity of the particle through the surrounding medium.

In order to calibrate the force with respect to movement of the particle or object, the trap stiffness of optical traps must be determined. One method is to move the entire mi-croscope stage with the particle on it as was done by Simmons and coworkers [19]. Such a movement creates a Stokes drag force which acts in opposition to the gradient force.

κ π η=x a v6

Thus the trap stiffness may be obtained by measurement of the stage velocity and particle displacement as long as the particle radius and fluid viscosity are known. Such an approach requires less actuator bandwidth than many other methods since measurements can be taken relatively slowly.

Fopt=−kx

Fdrag=γ0v where γ0=6πaη

kx=6πaηv

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2.2 EQUIPARTITIONAnother way of calibrating optical traps utilizes the equipartition theorem which states that in thermal equilibrium, energy should be evenly distributed in all of its

forms. The one-dimensional kinetic energy of the particle is k T1

2 B where kB is the

Boltzmann constant, T is the absolute temperature. The potential energy held in the

optical trap is given by κσ1

22 where σ2is the variance in the position of the particle.

Setting the two equations as equal and rearranging we get:

κσ

= k TB2

This allows the trap stiffness to be determined without any need for knowl-edge of the viscosity. This equation can then be applied in each dimension in which particle position can be measured. It should be noted that in order for this technique to be valid, a critical threshold of measurements is needed such that the variance and mean squared displacement of the particle converge to a constant ra-tio. Since this technique relies on accurate measurement of the variance, care must also be taken to avoid low-frequency noise due to laser instability, mechanical drift or thermal fluctuations which would give the appearance of a lower apparent trap stiffness [20].

2.3 LANGEVIN METHODA micron-sized particle held in an optical trap in an over damped media, such as water is often subject to thermal excitation through continuous bombardment by fast-moving molecules. If the particle is weakly held in the optical trap the particle’s movement traces out a constrained Brownian walk in a potential well. The Brownian motion is easily noticeable and can be detected by cameras and photodiodes. The thermal force takes the form:

π ηξ=F k Ta t12 ( )therm B

Here ξ(t) is a random Gaussian process, T is the absolute temperature, and kB is the Boltzmann constant. The thermal force can then be balanced against the gradient force, κ x t( ), the Stokes drag force, γ v( )0 t , and the inertial force to give the following Langevin equation:

γ κ γ ξ+ + =mx t v t x t k T t( ) ( ) ( ) 2 ( )B0 0

The inertial term mx t( ) can be dropped since it is negligible in a low Reynolds number media. Equations of this form have standard solutions. It can be shown that the time-dependent autocorrelation function of such an equation is given by:

κ=

κγx t x

k Te( ) (0) B

( )0

12kBT

12kσ2

k=kBTσ2

Ftherm=12πkBTaηξ(t)

kx (t)γ0v(t)

mx (t)+γ0v (t)+kx (t)=2kBTγ0ξ(t)

mx (t)

x(t)x(0)=kBTke(kγ0)

92 Measurement of position and force

This indicates the correlation between the position of a particle at an initial time and subsequent time t. By taking the Fourier transform of the autocorrelation func-tion, one can derive an expression for a power spectrum of the particle movement. The power spectrum takes the form of a Lorentzian curve.

π γ=

−S

k T

f f2 ( )xB

20 c

Fitting such a function allows the determination of trap stiffness. The corner fre-quency fc of such a curve is given by:

κπγ

=f2c

0

It is also possible to determine the trap stiffness using the equipartition theorem by integrating the area under the Lorentzian fit [21] (Fig. 1.3).

2.4 LIGHT DEFLECTION METHODThough most methods for calibration of the trap stiffness are indirect, it is possible to measure the force directly. As has been mentioned earlier, the force exerted on the particle is proportional to the deflection of the incident beam. Thus if the light

Sx=kBT2π2γ0(fc−f)

fc=k2πγ0

FIGURE 1.3 Power Spectral Density Plot of a 5 µm Silica Bead in Water Held by an Optical Trap

The smooth line is a fitted Lorentzian curve that can be used to accurately find the value of the trap stiffness

10 CHAPTER 1 Human gesture recognition for optical manipulation

deflected by the particle can be measured by a photodiode or position sensitive detector the momentum transfer to the trapped object can be directly calculated, as noted in [22–24]. This is advantageous since prior knowledge of the particle or the medium is not required. However, a well-engineered light collection system is required since such a technique requires full knowledge of all the light scattered by the particle.

3 SYSTEM DESIGN AND INSTRUMENTATION OF OPTICAL MANIPULATION SYSTEMSAutomation is ubiquitous in modern complex systems, such as vehicles, robots, and biomedical systems, and optical manipulation systems are no exception. Researchers and engineers often use control theory during the design of a system. In this section, we will try to cover some basic concepts in the design and implementation of opti-cal tweezers systems. These concepts are often overlooked in literature however are crucial to the realization of high quality optical trapping systems particularly those where robotic operations are required.

3.1 SYSTEM DESIGNThe design of modern complex systems is a process of selecting suitable devices and interfacing these devices in order to achieve desired performance. Most systems are built on vibration-isolated tables. With computers and wires either suspended above or below the table. The current trend is to use a dedicated, microprocessor-based control system for each device [25]. There are many commercial devices available that can be used directly in an optical manipulation system. Selection of the appropriate hardware components requires a basic knowledge of the parameters used to evaluate the equipment and how they affect the performance of a system [25,26].

For most optical manipulation systems, the main components are:

1. Microscope: Modern microscopes have become an indispensable part of modern research. Products from the major microscope manufactures, such as Nikon, Leica, Olympus, and Zeiss provide durable mechanical components, interchangeable modules, and computer controlled features. It is important to carefully compare the magnification, resolution, working distance, illumination, and imaging sources when choosing the microscope brand. Ultimately the limit on the resolution of any optical system is set by the diffraction of light; an optical system that performs to this level is termed “diffraction limited.” In this case, the spatial resolution is given by the Rayleigh criterion:

λ=dNA

0.61d=0.61λNA

113 System design and instrumentation of optical manipulation systems

Where λ is the wavelength of light being imaged by the microscope, d is the smallest resolvable distance between two points in the sample, and NA is the numerical aperture of the microscope objective. Similarly when a Gaussian laser beam with wavelength λ is used to trap a particle, 2d is the diameter of a focused laser spot at the focus.

In the case of commercial microscopes, a beam for optical trapping can often be inserted through a viewing or illumination port. This may, however, present difficulties in alignment or imaging. An alternative is to insert a thick dichroic mirror directly below the objective holder. This can then be coupled to an external optical train. In the past, this was done with custom made parts but now several microscope companies provide integration for such beams.

2. Computer: A powerful computer is the central part of many systems. Used to receive, save, and process signals from the detectors, cameras, and sensors that generate commands to both activate the actuators, and to communicate with controllers. Processing speed and storage capacity are key factors when choosing a computer. Another important factor to consider is the interfacing of hardware devices. For instance, many older hardware companies use PCI and PCIe bus lines to communicate with the computer but nowadays computer manufacturers only provide one PCIe slot and no PCI slots. Though laptops are replacing desktops in many work environments It is still comparatively difficult to interface many control systems to laptops. However, recently there has been a growth in USB control. So future systems may be increasingly portable.

3. Camera: Quality imaging is a critical component in the optical manipulation systems as it is used to detect particle motion and, by extension, force [27,28]. There are many digital cameras exclusively designed for microscopes, such as Hamamatsu, Photometrics, Nikon, Andor, Sony, Cohu. There are two factors to consider when selecting a digital microscope camera: sensitivity and resolution. The sensor is classified as either a CCD or a CMOS, both of which convert photons into electrical signals. The larger the sensor or the bigger the pixels are, the more sensitive it will be. The resolution is the total number of pixels that actually form the image. Make sure the camera resolution exceeds the resolution of the microscope’s optics by at least a factor of 2 (Nyquist limit). For force calibration or particle tracking applications, high-speed cameras may be necessary especially when multiple objects or robotic actuators must be tracked at once (see sensors). Several companies make high-speed cameras, such as AVT, Mikrotron, and Photron. Since the exposure time decreases as the frame rate increases, sufficiently strong illumination is also a prerequisite to prevent darkening of the image.

4. Actuators: The actuator is the device that mechanically drives a dynamic system. Actuating devices include stepper motors, DC motors, AC motors, solenoids, voice coil actuators, and relays [25]. Proper selection of actuators and their drive systems during the design process is very important. The parameters to select the best actuators are accuracy, sensitivity, noise, stability, bandwidth, speed, oscillation. Commonly used actuators in an optical manipulation system

12 CHAPTER 1 Human gesture recognition for optical manipulation

include microscope stages [25,29,30], sensor-based piezo actuators [25,31–34], rotary stages [30], piezo-mirrors [34], fast steering mirrors [30], SLMs [35–37], AOMs [19], and galvanometer scanning mirrors [38]. The most relevant specifications used in selecting actuators and their controllers are travel range, resolution, repeatability, stability, speed, computer interface, and compatibility between commercial software.

5. Sensors: A sensor is a device used to measure a given physical quantity [29]. Choosing the appropriate sensor depends on its sensitivity, resolution, bandwidth, measuring range, and dynamic responses. In optical manipulation systems sensors are commonly used to measure position and force. The force can be measured by a calibrated fast responding sensor. It is then possible to modulate the force by feedback with actuators [35].

The most commonly used sensors for force measurement are quadrant photodiode detectors (QPDs) and high-speed cameras:a. QPDs: These detectors consist of a photodiode divided into four identical

sectors. A photodiode makes use of the photovoltaic effect, in which a photon falling onto the sensing surface induces the release of an electron from a valence band. Photodiodes divided into four sectors can reveal the relative motion of a light beam in vertical and horizontal directions. By measuring the voltage differences between quadrants, the user can tell how the beam is moving as shown in Franck 2010 [33].

b. Cameras: High-speed cameras are reliable alternatives for the direct characterization of the optical trapping force and particle motion, replacing indirect motion measurements often performed by quadrant detectors [27]. Such applications require particle positions to be logged in the kiloHertz range and above. Measurement of the displacement of a particle from the trap center can also be related to the applied force [39]. Characterization using high-speed cameras relies on the proper estimation of the trapped particles displacement directly from an image sequence [20].

3.2 SYSTEM IMPLEMENTATIONAfter carefully selecting all the hardware components, the next step is to build a control system. Control systems can be used both to control the hardware of the opti-cal trapping system, for instance, the microscope stage, and also they are often nec-essary for the control of trapped objects themselves. This is particularly necessary if the particles must be trapped without direct user intervention, such as in the case of fast swimming bacteria or sperm or if the trapping is to be an entirely automated task. The computer is the center of real-time operations where processing has to be synchronized with operation and actuation requirements [25]. Most of the com-ponents can be controlled by a computer through Serial port, IEEE1394, Ethernet, FireWire, USB ports, PCI (peripheral component interconnect) bus, or PCIe bus. In some cases, modern computers without serial ports may require serial-to-USB con-verters to allow compatibility with RS 232 serial devices. A typical block diagram

133 System design and instrumentation of optical manipulation systems

of an optical manipulation system using a computer as the central piece to control all the hardware components can be illustrated as shown in Fig. 1.4.

The next step in the implementation is to computer control each of the actuators. Some actuators in the optical manipulation systems use feedback control architec-ture, such as the servo microscope stage controllers (ASI, LUDL et al.), sensor-based piezo actuators [25,26], which may require the fine tuning of the parameters to achieve the best speed and accuracy. This can also apply to optically controlled actuators, such as photonic force microscopes (mentioned later in the chapter).

In a feedback control mode, shown in Fig. 1.5, the control loop has to be closed by measuring the system response and employing that information to generate control sig-nals to correct any output errors [25]. There are various control modes, such as on/off, proportional (P), integral (I), and derivative (D) or combinations in the controller box. Proportional-Integral-Derivative (PID) control is the most common control algorithm, the integral of the error over a time interval and the rate of change of the error is used to determine how much of a correction to apply by a weighted sum:

∫= + +u K e K edt Kde

dtp i d

where u is the control signal, e is the error signal, Kp is the proportional gain, Ki is the integral gain, and Kd is the derivative gain.

u=Kpe+Ki∫e dt+Kddedt

FIGURE 1.4 Block Diagram of the Hardware used for an Optical Manipulation System

FIGURE 1.5 Feedback Control

14 CHAPTER 1 Human gesture recognition for optical manipulation

Tuning the PID parameters is essential to achieve the optimal performance of the actuator. For most cases, the optimal performance refers to the best combination of three factors: less overshoot, fast rise time, and short settling time. The tradeoff of each parameter is:

1. The proportional control mode is the main driving force in a controller to provide the necessary speed of response and adequate signal level, but the shortcoming of increased proportional action is the degradation of stability.

2. The derivative control mode produces an output based on the rate of change of the error, which ensures stability. But the shortcomings of the derivative are insensitive to the steady state error, and amplifying the high-frequency disturbance inputs and noise.

When an error presents, the integral control mode will continuously increment or decrement the controller’s output to reduce the error, including small errors that persist over extended times, for example, the steady-state error. The shortcoming of the integral action is its slow response to sudden changes (Fig. 1.5).

For some optical manipulation systems, incremental-drive actuators or pulse-driven devices use the open-loop control mode to achieve position control, such as the stepper motors for short and repetitive motions at relatively low speeds [25,29,30], the piezo actuators which are simple, robust to modeling error, and effectively reduce piezoelectric nonlinearity at low-frequencies [27,28]. So-called “open-loop control” as shown in Fig. 1.6, is defined by the fact that the output signal is neither measured nor “fed back” for comparison with the input signal. In other words, the control vari-able adjustment is not error-based, it “assumes” that the desired goal of the input is successful. Open-loop control is used mostly in systems where the controlled vari-able behavior is well understood, for example, by modeling and/or prior calibration, and not influenced by any outside disturbances. The advantages of using open loop control are: simple in construction and design, economical, easy to maintain, gener-ally stable, convenient to use when output is difficult to measure. The disadvantages are: inaccurate and unreliable under unexpected perturbations. It is therefore recom-mended to check the accuracy of those actuators periodically using another type of measurement or feedback.

In this section, a real-time automated tracking and trapping system (RATTS) [40] is chosen as an example to illustrate the procedure for the design and implementation of an optical manipulation system outlined earlier. The main goal of the RATTS ap-plication is to use optical tweezers to measure sperm motility and to quantitatively assess differences between species. The first step in the system design was to carefully select the components to achieve those goals. The key components of this optical manipulation system were chosen be to: (1) a microscope (Zeiss) to view the sperm

FIGURE 1.6 Open-Loop Control

154 Human interfaces

sample and the laser trap, (2) a host computer with 2 PCI slots and two hard drives with RAID level 0 controller configured for data striping, (3) a CCD camera (Sony) with 30+ frame/s rate for real-time imaging; it also served as the sensor for the laser trap location, (4) a laser source: near-infrared continuous wave 1064 nm wavelength laser (Nd:YVO4), (5) a motorized microscope stage (LUDL) to keep the sperm of interest in the field of view, (6) a linear polarizer in a rotary stepper motor mount (PR50PP) to control the trapping power precisely, and (7) a mechanical shutter (Unib-litz) in the laser path to turn the trap on/off to trap or release the sperm of interest.

An image acquisition board (National Instruments, NI, PCI-1409) was used to connect the CCD camera to the computer. A motion control board (NI, MID-7604) driven by PCI 7358 was chosen to control the XY microscope stage controller and the rotary stepper motor. The mechanical shutter can be controlled through two lines of digital input–output from the motion controller.

For fine-tuning the parameters of the two actuators: microscope stage and the rotary stepper motor; the encoder mounted on the microscope stage can serve as the feedback for the stage controller and the PID parameters can be tuned using NI MAX interface software. The microscope stage can also work in an open-loop mode, and the camera was used as a sensor to double check if the stage moved to the desired position. To calibrate the system a micro ruler was placed on the microscope slide. The system acquired images of the micro ruler before and after the stage movement. In this way one can clearly determine the accuracy of the stage control when working in the open-loop mode. A rotary stepper motor was used in conjunction with a linear polarizer to change the laser trapping power; a power meter can be used to check the accuracy of its movement before and after the laser trapping experiments. This avoids hysteresis as the power readings can be compared with the previous records to know if the rotary stepper motor moved to the desired position.

Having discussed basics of the theory, implementation, and low-level control of optical tweezers systems, we now turn the reader’s attention to the higher-level com-puter control and human–computer interfaces used in such systems. We will devote the rest of the chapter to this subject.

4 HUMAN INTERFACES4.1 SOFTWARE CONTROL OF OPTICAL MANIPULATION SYSTEMSAfter connecting the hardware components to the computer, and fine-tuning the pa-rameters of the actuators, the next step is to choose a computer language to collect the data from the sensors, save and process the data real-time, generate the com-mands to control the actuators. Presently, engineers, researchers, and scientists use advanced software suites for their unique applications. Various research laboratories use different software platforms to develop toolkits or modules and some are made available for other researchers and engineers working in the optical manipulator field to freely download: IDL was used for particle tracking and holographic optical

16 CHAPTER 1 Human gesture recognition for optical manipulation

trapping [28], OpenGL to calculate holograms on the graphics card [41], MATLAB to write a tweezercalib toolbox for precise calibration of optical tweezers [42], and to calculate the optical forces and torques [43], and LabVIEW for a fast hologram generation and particle tracking [44].

In cases where the optical manipulation system requires a special operation or the free or commercial software packages does not support a particular hardware compo-nent, the researchers need to write a custom-designed program to meet these special requirements. Comparing with other commonly used software platforms, LabVIEW allows the researchers to spend less time addressing text-based syntax and more time solving complex system challenges. Many research groups have chosen LabVIEW for their particular applications to reduce the testing and development time, such as the instrumentation control and data acquisition to study of kinetochore-microtubule interactions [33], for real-time evaluation of red blood cell elasticity [32], for the design of a scanning laser optical trap for multiparticle manipulation [45].

In this section, RATTS [40] is also used as the example to illustrate software con-trol in a typical optical manipulation system. In order to achieve the goal of a quantita-tive measurement of sperm motility, an automated software program to measure both the swimming force and the swimming speed of individual sperm was developed. The flow diagram for software control is: (1) a user picks a sperm of interest from acquired sperm images and clicks to perform the image processing algorithm which identifies the location of the sperm and to calculates its swimming speed over time; (2) the system traps the sperm of interest by moving the microscope stage and then slowly reduces the trapping power to avoid photodamage and to allow the sperm to escape once the sperm is in the laser trap; (3) the system detects the moment a sperm escapes the laser trap and calculates the trapping power at which it escaped, which is the indicator for the sperm swimming force. For this time, sensitive hardware inten-sive application, LabVIEW was chosen to be the software platform since it provides a single programming interface to all data acquisition devices, resulting in seamless hardware and software integration. The flowchart of RATTS software is shown in Fig. 1.7. The image acquisition, enhancement, segmentation, collision detection were discussed in [46] and the video rate tracking and trapping are discussed in [40].

5 CONTROL WITH PERIPHERAL DEVICESRecently, there has been growing use of external peripheral equipment to control nanorobotic systems. Such devices can be used in place of a standard mouse and keyboard. After appropriate engineering and calibration, many micro- or nanooptical systems can be controlled almost exclusively via computer. Then it is easy to connect a computer to a whole host of external devices that may be controlled by the user.

Perhaps one reason behind this growth in popularity is the parallel advance-ments in both computer control and display technology. Several companies, such as Microsoft, Sony, Samsung, and others have launched products aimed at improving the user’s multisensory experience. A decade ago computer companies eschewed

175 Control with peripheral devices

research into external devices in favor of improved graphics and ease of use for their customers. Today, there is a resurgence in new devices aimed at all areas of the marketplace from virtual reality headsets to augmented reality capable smart phones, which add new levels of user interaction into the computing experience. But why might researchers interested in nanorobotics be interested in these devices?

There are several reasons:

Enhanced user experience and ease of use:External devices, such as joysticks, steering wheels, pointers, guns etc., offer a way to facilitate interactions that otherwise might not be possible or might be

FIGURE 1.7 Flowchart of the Software Control of RATTS

User input was limited to set parameters prior to an experiment and selecting, via the computer mouse, a sperm in the field of view for analysis. The mouse cursor coordinates were registered and passed to the tracking algorithm, and computation proceeded with no further intervention. Once the specified numbers of frames have been processed, the stage was moved to place the sperm under the laser trap whereupon the shutter was opened.

18 CHAPTER 1 Human gesture recognition for optical manipulation

difficult using a standard mouse and keyboard setup. Users can become part of a human machine feedback loop where complex signals can be transmitted and received faster, more naturally and more intuitively. By improving the ease of use of a device the efficiency of the device itself is improved. The user is also freed to think “what to do” rather than “how to do it.”Extra information received by the user:In some cases, extra information can be displayed to the user with peripheral devices. This can range from auditory cues, vibrations, depth information, or even information about textures or forces. Examples of this are found in Haptics and augmented reality (AR)/virtual reality(VR).Extra information is given by the user:Sometimes it is desirable that the user can impart instructions to control a device or probe. Voice commands and gestures can be an easy way to do this. In order to impart spatial information, 3D motion tracking can be used to capture user gestures or actions. This can also be beneficial where high-speed user decisions must be taken that would otherwise take too much time to be specified via a keyboard, etc. 3D motion controllers are used in computer game controls and even in fitness bracelets.Demonstrations or collaborative working:A strong reason for creating a device that can be used by multiple users is to allow a collaborative working experience [47]. This can help draw together experts from different disciplines to work on a particular problem or sample or can be used to train novice users. Such interactions can be created in the same room or remotely from different locations[30].Specialist tools:Some micro tools, such as probe devices or photonic force microscopes may not lend themselves to mouse and keyboard control. In this case, specialized control equipment may better allow the user to accomplish tasks quickly and efficiently without the need to subdivide commands into keyboard controlled sequence [48,49].

One of the most effective and simple additions to optical trapping technologies has been the multitouch environment. Since optical trapping experiments often re-quire simultaneous control of multiple particles or objects, a platform that allows the user this control is beneficial. [47,50] Unlike the standard PC, where operations are largely serial in nature, multitouch devices allow multiple movements that simplify the user experience.

Initial experiments with finger tracking used white pins attached to gloves. The white pins could be tracked in three dimensions by a camera [51]. The position data was then used to create a series of dynamic optical traps. Grieve and coworkers [52] used a multitouch table to facilitate a collaborative working environment. Images from the computers’ display were projected onto the bottom of an opaque viewing table. Frustrated total internal reflection of light produced by 880 nm LEDs was used

196 3D control

to track the positions of the user’s fingers [53]. This was then captured via CCD and processed computationally.

The system was also used to control high-speed AFM enabling the user control over the scan area [54] (Fig. 1.8).

Bowman and coworkers [55] has used the Apple iPad to control multiple opti-cally trapped particles. This allows the user to simultaneously manipulate several particles independently rather than one at a time or together as a group. In this case, a host computer controls a Zeiss microscope setup and relays images to the iPad via Wi-Fi. The positions of the user’s fingers are recorded via Apple’s gesture recogni-tion toolkit and then sent back to the host computer. This approach allows the user the flexibility to move away from the laboratory and enhances user experience.

6 3D CONTROLOne challenging aspect of working at the micron scale and below is the ability to access spatial information in an intuitive way. Conventionally, most researchers will be familiar with looking down a microscope at a sample. Unfortunately, this view of the world often gives insufficient or distorted information about the out of plane aspects of the sample, which is crucial information for the movement of nanorobots in 3D. In order to facilitate adequate control or navigation of nanotools informa-tion about the 3D environment surrounding the tool must be gathered before being processed and displayed. After processing, instructions can be sent back to actuators

FIGURE 1.8 The Multitouch Console in Action, Users Interact With the System to Reposition Groups of SiO2 Spheres

20 CHAPTER 1 Human gesture recognition for optical manipulation

moving the tool [56]. Both the “sending” and “receiving” aspects of this operation can present substantial difficulties.

6.1 GATHERING SPATIAL INFORMATIONTo gather appropriate spatial information, a variety of techniques may be used. Most simply, 3D information can be collected via orthogonally orientated micro-scope objectives [8,57], allowing the user to see both a top and a side view. This can be extended to a top, side, and front view if required. In many situations, however, space limitations preclude the use of extra objective lenses. In this case, alternative techniques may be employed. When objects are transparent, the scattered light they create can be analyzed and depth information recovered. A simple example of this is the analysis of the Mie scattering pattern from a suspended polystyrene sphere in water.

If the pattern of rings produced by coherent illumination is analyzed by look-ing at the fringe spacing, depth information related to the position of the sphere relative to the focal plane can be derived [58]. Perhaps surprisingly, this technique also can be applied in the case of white light illumination albeit over a more lim-ited range of depth [28,59]. Holographic microscopy uses a similar approach to obtain depth information. In this case, information about the entire field of view can be derived.

Another approach is to use stereoscopic imaging [60], in which a single micro-scope objective is used, but the incoming light is sorted according to the direction it entered the objective. In this way, two images can be created taken from different angles. Correlation of these images can yield depth information. Depth information can also be obtained by using tomographic techniques, by scanning the focus of a microscope objective, or from color information contained within images [61–64].

6.2 SUPPLYING 3D INFORMATIONThe 3D control of the objects also presents difficulties. If the nanotool is computer controlled, then a substantial amount of control theory may be required, as men-tioned earlier in this chapter. If the user is in direct control of objects on the slide, then a method for capturing spatial information from the user is required.

In recent years several peripheral devices, which allow the user to collect depth information, have become available. Many of these were developed for use in the computer games industry but have since found use in other areas.

A great example of this is the Microsoft Kinect, initially developed as an acces-sory for Microsoft’s X-box operating system. The Kinect provided a cheap and ac-cessible 3D sensing system to the general public. Within months of its release, there were several user-generated development kits and tutorials for how to use the Kinect on a standard PC and official development kits eventually followed. Apart from the Kinect, a number of similar systems have been utilized for tracking user gestures in 3D, such as the Sony Playstation Move and NintendoWii Motion Plus.

216 3D control

In the case of the Kinect, the depth-sensing camera can estimate distances of objects by continuously projecting and interpreting a pattern of spots from an IR light source. This allows the Kinect to acquire 3D positional information at a frame rate of 30 fps [65,66]. This data can then be fitted to human movement via 3D skeletonization algo-rithms. In practice, this means that a user’s body positions can be interpreted as a set of spatial coordinates. These coordinates can then be scaled and translated to positions in the spatial domain of the nanotool [49,67,68]. Fig. 1.9 shows how this can be applied in practice. With the use of the sophisticated “LCARS” software developed in [67] parti-cles can be moved, grouped, translated, scaled, and rotated. This allows the user to have control over two particles in isolation (one per hand) or multiple particles as group. Such a system allows single particle control but also allows multiparticle experiments too.

Recently devices which more specifically track hand movements have become available, such as the leap motion device [69–71]. This allows acquisition of 3D coordinates from finger movements rather than arm movements and continues the trend toward systems with fully integrated motion tracking.

With the increasing diversity of 3D tracking technologies, it has also become possible to display data to the user in 3D. Fig. 1.10 shows the author’s head being tracked by the Kinect system. While simultaneously the system displays a 3D model of the motion of 5 µm silica particles undergoing Brownian motion. As the user’s head moves, the display adjusts to show the particles from different perspectives giving a pseudo 3D display.

Similar ideas have been used to display data or to aid perception, such as eye tracking [72], which identifies the position of the observed particle and uses it to autofocus optical tweezers onto that point.

With the ongoing ubiquity of 3D sensing technology, it may soon be a matter of course for all computer interactions to be made as gestures. Such an approach lends itself to micromanipulation. As ever, the utility of such technology is dictated by the skill of its implementation.

FIGURE 1.9 The Kinect System Tracks the Hand Movements of a User Controlling 4 Particles Simultaneously

Gestures are used to separate one particle from the group and move it independently.

22 CHAPTER 1 Human gesture recognition for optical manipulation

7 HAPTICSAlthough it is useful for users to see how objects move at the micron level in real time and to manipulate them in a natural way, this misses out on another source of information: the sense of touch. Haptic interfaces allow the user kinesthetic com-munication with either a computer or an outside source of information. Such an approach can be used to convey information about the microenvironment directly to the user by adding a new dimension to the interaction [73].

Haptic tools have been in service for several decades [74]. Early devices centered on remote operation of robots for use in handling dangerous substances, such as in nuclear fuel rods or for use in aeronautics or military applications [75,76]. However, the idea of telepresence (the use of virtual reality technology for participation in remote/distant events) has been appealing to those wishing to delve into microscopic interactions for some time too [77]. Haptic feedback has become familiar to many of us in the form of vibrations emanating from video game controllers.

In its most simple form, the haptic interface may consist of a simple vibratory stimulus designed to prompt the user after an event. This can be useful, for instance, to avoid user error or to act as a warning or signal. However, this approach somewhat

FIGURE 1.10 The Author Using a Head Tracking Algorithm to Create a Pseudo 3D Display

A monitor (right) displays a 3D model of the motion of 5 µm silica particles undergoing Brownian motion. Images of the particles are taken by a high-speed camera (Prosillica GE680) and tracked in real time. As the user’s head is moved (A) right, (B) middle, and (C) left the display adjusts the vanishing point of the image accordingly. Giving the impression of a 3-dimensional image. Here a white spot denotes the particle’s 3D position, while the image of the particle is shown as a plane below the particle.

237 Haptics

sells haptic short. Haptic interfaces can also be used to convey sensory information, such as surface roughness [78] as well as information about nanoscale forces and can be coupled to both AFM [79] and photonic force microscopes [80,81]. It is worth noting that the human body does not perceive visual signals much above the range of 24–30 Hz as opposed to the sense of touch at 1 kHz [82], the haptic interface thus allows faster response and interaction.

In order for the users to both control and respond to interactions mediated through either a microtool/particle a “haptic coupling loop” is necessary [83]. This bilateral process [84] allows the user control of a robotic system that relays positional in-formation to another system actuating the nanobot/tool. This, in turn, sends back information about environmental interactions to the first system, which is then sensed by the user.

In order to practically create a haptic interface for microtools, however, there are several important factors that must be taken into account. First, there is the difference in the expectation of the user about how the world should function and the actual forces present in the micro/nano environment. Conventionally, we are used to an environment where inertial forces are dominant over viscous forces. However, in the microscopic world, viscous forces dominate [85]. Frictional, electrostatic, optical, and hydrodynamic forces may also play a role in the motion of objects. This risks undermining the intuitive nature of the interface. Though this may still provide an in-teresting user experience, there are also practical difficulties. Creating a system with direct coupling between the macro and microenvironments can result in stability issues. Most obviously, random vibrations introduced into the system by Brownian motion are then amplified by inertia at the macroscopic level. In order to combat this, it may be necessary to introduce additional damping to improve stability.

Another important factor to consider is the perceived distortion of the world by scaling factors. Let us consider a 5 µm latex particle moving under Brownian motion. Whereas a displacement of 1 µm can be scaled by 104 to a 1 cm movement percep-tible to the user, the force on the particle from Brownian motion must be scaled by approximately 1012 to generate roughly a Newton of force on the user’s hand.

Such distortion renders the system sensitive to perturbations and to delays and increases the effects of viscosity relative to weight, for instance [86].

In order for such coupled systems to work well, they must also have a high enough data transmission rate to be responsive and have low enough lag times so that instabilities in the haptic coupling loop are not created. In order to accomplish this, it is necessary to have fast enough sensors and actuation. Luckily, several technolo-gies for sensing and actuating exist that satisfy this requirement. In optical tweezers systems, particle displacements and forces can be measured at up to 10,000 fps with cameras and up to several megahertz with quadrant photodiodes [20,87,88]. Force sensitivity can be increased by force feedback, which also serves to mitigate the effects of random Brownian oscillations [35].

Actuation of particles can be achieved at similar rates by utilizing acousto-optic deflectors at 30 MHz [73]. Actuation response is limited in practice by drag forces, which limits actuation above the characteristic frequency of the particle [89].

24 CHAPTER 1 Human gesture recognition for optical manipulation

Computer processors can process signals at GHz frequencies but these signals can be delayed in practice by slower computer architectures, bus speed, cabling etc. or by inefficient high-level software implementation. Many control tasks may also be distributed to chip-based systems, such as FPGA’s which although difficult to program can respond faster than standard PC's.

8 INTERNET CONTROL—CONTROLLING SYSTEMS REMOTELYThe Internet has not only changed the way scientists and engineers do research and development, but has also reduced many barriers regarding collaborations. WiFi controlled technological devices become part of everyday life. With fast expanding Internet usage, researchers should be able to conduct experiments in a cost-effective and time-efficient way. Web-based research servers can provide a platform for interactive virtual scientific collaboration and can carry the concept of “a shared technology resource.” This leverages sophisticated state-of-the-art technologies so they are generally available to the scientific community thus, reducing the need for duplication of resources and allowing researchers to conduct feasibility studies be-fore investing in a similar (or scaled down) technology.

Researchers at the Universities of Glasgow and Bristol, UK, developed an Apple iPad application, iTweezers, to control optical tweezers, which can be op-erated remotely through Internet [42]. The app streams images over WiFi from a computer-controlled optical tweezers system. Users can operate up to 11 optical traps on screen at a time to grab and to manipulate multiple particles simultane-ously. This iOS app is freely available to download and can run Glasgow’s free Red Tweezers software to control any optical trapping system remotely outside of the laboratory.

Two biophotonics laboratories at the University of California, San Diego (UCSD) and the University of California, Irvine (UCI) have implemented a web-based re-search service through the LOGMEIN platform for the last decade [30]. They have provided the broad biomedical scientific community “one-click” access to any of eight the robotic laser-manipulation microscopes (RoboLase).

In this system, the optical tweezers are split into two beams using a polarizing beam splitter, which creates two “hands” that can trap or “hold” microscopic ob-jects [90]. After these objects are trapped, the individual positions of the S polarized trap and the P polarized trap can be controlled and thus the held microscopic objects can be moved anywhere the user desires. Optical scissors, whose position can also be controlled individually and simultaneously, can perform precision laser cutting in any shape and location within the field of view. Thus, simultaneous optical trap-ping and cutting make optical micromanipulation of microscopic objects possible. This capability is analogous to having a set of two microscopic hands and one pair of microscopic scissors that can be used to manipulate objects viewed under a mi-croscope.

258 Internet control—controlling systems remotely

Depending upon available bandwidth and data compression rates, all collabora-tors are able to observe the biological, chemical, or physical responses of the samples by viewing the images displayed on the website from the host system in “internet-real-time.” Collaborators can then discuss the observations and results through an on-line chat window and request remote system-control at any time during the experiment. The RoboLase systems have been used to cut single 25 nm diameter microtubule [91], to make 80 nm cuts in single chromosomes [92], to ablate a 100 nm diameter centriole in cancer cells [93], to generate a double strand break and study the roles of different protein in homologous recombination (HR)–mediated DNA double-stranded break repair [94,95].

Web-based research not only allows for scientists to participate in the research from remote areas but also stimulates and facilitates multidisciplinary team collabo-rations via sharing resources and knowledge (Figs. 1.11 and 1.12).

FIGURE 1.11 Structure of an Internet-Accessible Platform for Interactive Collaboration

26 CHAPTER 1 Human gesture recognition for optical manipulation

9 FUTURE DIRECTIONSWe are currently enjoying the dawning of a new age of human-machine interaction. Though we do not now enjoy the symbiotic relationship of the cyborgs of science fic-tion, it is now possible to interact directly with distant machines, remotely controlled vehicles, and robots. Modern technology has enabled interactions with machines, which is both intuitive and natural. This technology presents exciting possibilities for scientists interested in discovering new phenomena. With greater freedom to ex-plore, research is made easier and quicker. But such technology is likely to present important challenges.

Optical nanorobotics is, in some ways, far behind in macroscopic companion in terms of the design of individual parts, fabrication, and manufacturing of compo-nents. There is also little standardization of equipment or approach. This means that fully active micromechanical systems are for the most part still limited to the labo-ratory. This is not to say, however, that useful systems cannot be developed today. Indeed, the various useful examples in this book paint a promising picture.

As for human interfaces, many new technologies are around the corner that will aid human-machine interaction. Several systems involving haptics, virtual reality, and 3D control are currently in development. Commercially, virtual reality is a fast-growing sector. Sony, Samsung, Google, Microsoft, and HTC are all develop-ing new virtual reality technology for release in 2016/2017. We can look forward to higher refresh rates and resolution and low persistence and lag times in such devices.

Focused ultrasound has been used to create haptic feedback in mid-air or above a surface [96]. This has the potential to be combined with augmented reality to make

FIGURE 1.12 Front Panel Displayed on the Screen of RoboLase IV (UCSD)

27References

a highly effective three-dimensional working environment. Crucially, it could be ac-cessible to multiple users which may fair better in a laboratory environment.

In the last few years, new technologies have become available which control ro-botic interaction via a direct brain link [97]. This could signal plausible mind control of robotic systems [98,99]. Such systems could obviate the need for any dexterous interaction whatsoever.

As Internet based resources continue to grow, we can look forward to more dis-tributed experimentation [100], allowing laboratories worldwide to share expertise and resources more effectively. Virtual spaces for research could also be created for experimentation enabling students to learn technical skills before actually accessing experimental equipment. WiFi controlled medical nanorobotics could also lead to novel ways of accessing small spaces. Montgomery and coworkers[101] developed easy-to-construct implantable wireless optogenetic devices. In 2015, they reported the smallest version (20 mg, 10 mm3) of a wireless optogenetic system, which is two orders of magnitude smaller than previously reported. With a radio-frequency (RF) power source and controller, this implant was used to demonstrate wireless optoge-netic control of brain, spinal, and peripheral circuits in mice. It is easy to see how photonic devices could easily be controlled without the need for a central computer system but could be connected directly to a network.

As we have seen, interacting with objects at small scales can only be done in a nonintuitive way due to the scaling of optical, electrostatic, and viscous forces.

This means that any full implementation of a nanorobotic avatar or telepresence system would require a software based rescaling or augmentation of forces to enable intuitive or even practical interaction. This creates a "Hulk” paradox. How can a user be given super powers outside his native understanding without misconstruing or destroying things around him unwittingly? Deciding on the basis for this rescaling is in some sense contentious as it is necessarily a distortion of reality and therefore impedes understanding. Clearly, full-scale immersive experiences will require a sig-nificant amount of research and software development before they reach their zenith.

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33

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00002-1Copyright © 2017 Elsevier Ltd. All rights reserved.

Andreas Ostendorf, Jannis Köhler, Sarah I. Ksouri, Gordon Zyla, Cemal EsenApplied Laser Technologies, Ruhr-Universität Bochum, Universitätsstraße, Bochum, Germany

CHAPTER OUTLINE

1 Introduction in Microfluidics ................................................................................ 331.1 Definition, Materials, and Manufacturing ............................................341.2 Light-Based Microfluidics .................................................................351.3 Assembling of Microstructures ..........................................................361.4 Contents .........................................................................................37

2 Generation of Microstructures with Two-Photon Polymerization ............................. 373 Assembling Techniques ....................................................................................... 43

3.1 Chemical Bonding ............................................................................453.2 Thermal and Photothermal Connection ...............................................463.3 Joining by Polymerization .................................................................483.4 Interlocking Connection ....................................................................49

4 Applications for Assembled Microstructures ......................................................... 534.1 Optically Controlled Valves ................................................................534.2 Magnetic Microrotor: Flow Field Determination and Pumping ...............544.3 Microrotor Assembly Using Screw Connection .....................................57

5 Conclusion and Outlook ....................................................................................... 58References .................................................................................................................59

1 INTRODUCTION IN MICROFLUIDICSMicrofluidic chips enable complete laboratory analysis and diagnostics from smallest volumes of liquids on dimensions not exceeding a matchbox. In the process, these systems are based on integration and miniaturization of pumps, mixers, and devices to control the fluid flow. Light plays an important role for manufacturing and actuation of these microdevices and can be applied for further improving microsystems with re-gard to new capabilities and higher performance density. In the following paragraphs, an introduction to microfluidics, optofluidics, and assembling techniques is given.

Laser-based assembler and microfluidic applications

2

34 CHAPTER 2 Laser-based assembler and microfluidic applications

1.1 DEFINITION, MATERIALS, AND MANUFACTURINGThe processing and manipulation of liquid fluids in channels with dimensions in the micrometer range is referred to as microfluidics [1]. These channels enable low amount of liquid samples and reagents to separate, intermix, interact, or further pro-cessing. Additionally, the fluids and their ingredients and products can precisely be characterized and analyzed due to integrated technology. Hence, microfluidic sys-tems, also known as laboratory-on-a-chip (LOC) or micro total analysis systems (µTAS), have fast become a key instrument for analysis and diagnostics within sci-entific as well as commercial interest. Important contributory factors are fast reaction times and low waste generation due to the very small quantities of liquid flow. More-over, microchannel networks allow multiparallel analysis and complete laboratory protocols can be performed on one small chip, which usually covers an area of only a few square centimeters. Therefore, costs can certainly be reduced as well.

The areas of application range from biotechnology, medicine, and pharmacol-ogy, to food and environmental diagnostics, and many more. The multidisciplinary field of use and the growing interest have led to a variety of suitable materials as well as applicable production and manufacturing techniques. Especially polymers, for example, poly(dimethylsiloxane) (PDMS), have emerged as preferred substrate materials, because of advantageous chemical, mechanical, and optical properties [2]. Furthermore, low cost and easy handling contribute to the success.

To fabricate prototypes or small-scale series of microfluidic devices with high precision, microstereolithography processes can be utilized. Two-photon polymer-ization (2PP or TPP), for instance, has emerged as a powerful tool to manufacture three-dimensional polymer devices with submicrometer resolution [4]. In the process, a pulsed laser beam is focused into photosensitive material and leads to chemical re-action and polymerization within the focal point. Devices with desired geometry can, therefore, be generated line by line and layer by layer. Furthermore, the integration of different functions by embedding nanomaterials into the photosensitive material is feasible [5,6]. Customizations of the refractive index of the polymer, electrical con-ductivity, or chemical functionalization are achievable. Furthermore, by embedding magnetic nanoparticles, free standing microstructures can be controlled and moved by an external magnetic field. This can be applied for magnetic rotors or microtur-bines to displace surrounding fluid in a microchannel [7].

To transport and control fluids through microchannels and microreactors on a microfluidic chip, it is required to apply and integrate miniaturized pumps, valves or switches, and techniques for intermixing different fluids. Due to the small dimen-sions of the microchannels and resulting scaling effects, the fluid flow can be de-scribed by a low Reynolds number (Re) and a laminar flow is dominant. Thus, mix-ing only occurs by diffusion at the interface of the fluids and is ineffective and slow. In order to accelerate the mixing process, two different methods can be applied: First, passive mixers induce a chaotic advection by adjusted microchannel geometry, for example, staggered-herringbone grooves or zigzag-shaped channels are utilized [3]. Thereby, the mechanism of effective mixing is based on repetitive stretching and

351 Introduction in microfluidics

folding of the fluid flow. Second, active micromixers can be utilized for even faster and entire mixing of different fluids. Therefore, rotational or translational motion of microstructures, for example, rotors or turbines, is essential to induce perturbations. Magnetic, electrostatic, or optical forces can be applied to move the microdevices with desired rotational speed and direction. Furthermore, rotational and translational motion can be used for pumping and transporting fluid through the microchannels or for controlling the fluid direction by opening or closing certain microchannels.

1.2 LIGHT-BASED MICROFLUIDICSIntegrated techniques for sensing and actuation with laser light play an important and extensive role on microfluidic chips [8,9]. Such light-based devices are also known as integrated “optofluidics.” Optical forces, for instance, can be used for contactless trapping, moving, and applying torque to microobjects in so-called optical tweezers (OT) and are sufficient for different microfluidic applications where pumps, mixers, or valves are required [10].

Arthur Ashkin [11] first experimentally investigated the effects of radiation pres-sure on dielectric spherical microparticles in 1970. Refraction and reflection of a focused laser beam at the interface of the particle and the surrounding medium lead to momentum transfer between photons and matter. Arising optical forces can be divided into two components: First, the scattering force, which pushes the particle in the direction of light propagation, and second, the gradient force, which is directed toward the intensity maximum at the focal point. By using a microscope objective with high numerical aperture (NA), the gradient force can compensate the scattering force and a microparticle can stably be trapped at the focal point [12]. Furthermore, this particle can be moved in all three dimensions. The trapping forces acting on a particle are typically in the range of 1 pN per 10 mW [13]. Sophisticated OTs provide trapping of multiple particles at the same time and moving these individually. For multiplexing the beam and to obtain multiple tweezers, different techniques can be utilized. The light path can be split into two separate beams, which are controlled in-dividually by two steering mirrors and recombined before entering the focusing ob-jective [14]. Another method uses steerable mirrors to rapidly scan between several positions and allow a time-sharing trap configuration [15,16]. With the integration of an acousto-optic deflector (AOD) or electro-optic deflector (EOD) higher frequen-cies and, in consequence, more traps can be generated [17,18]. Holographic optical tweezers (HOT) make use of spatial light modulators (SLM), which enable desired phase modulation of the incident laser beam by electronically addressable liquid crystals [19]. Therefore, computer generated holograms can be used to produce the desired number of trapping spots and intensity distributions. Furthermore, the gen-eration of complex beam shapes, for example, optical vortices or Bessel beams, al-lows novel features and applications.

For the use on microfluidic chips, in particular for biological applications, the OT provides numerous advantages. The contactless trapping and moving of microobjects allow minimally invasive and proper handling with sensitive materials and biological

36 CHAPTER 2 Laser-based assembler and microfluidic applications

samples. As cells or other organisms are only transparent for certain spectral widths, absorption can be neglected with the use of carefully selected wavelengths. There-fore, trapping with the correct laser system can reduce heat generation to a minimum. Furthermore, the OT is highly versatile and multifunctional applications can be ac-complished at the same time. Besides actuation, systems based on optical forces can also be used for sensing and characterization. Microrheology, for instance, is a typical application where fluid properties are investigated, for example, viscosities or flow fields are measured [21–23]. Thereby, sensing with high detection accuracy and verifiability is realizable. For this very reason Padgett and coworkers [24] identify the OT as a technology with high potential for microfluidic applications. In addition, optical trapping can be achieved through other (semi-)transparent material [25] and with the aid of a SLM, focusing a laser beam through turbid and diffusive media is possible [26]. Finally, parallel manipulation, treatment, and detection with laser light and optical forces on a single microfluidic chip is feasible [20]. Zhang and coworkers [27] give an overview of recent advancements of the OT for applications regarding single cells.

1.3 ASSEMBLING OF MICROSTRUCTURESIn order to manufacture microfluidic devices, different methods have been inves-tigated. Assembling techniques, for instance, provide joining of multiple tiny components or building blocks to manufacture complex microstructures. One major advantage of assembling techniques on the micrometer scale is the combination of different materials within hybrid microstructures. The integration of various materi-als can lead to new metamaterials or structures with tailored properties, for example, mechanical, optical, or electrical properties and functionalization can be achieved as desired. Biohybrid microrobots can be assembled by using synthetic and biological components as discussed in Chapter 14, for instance. In addition, assembling tech-niques are appropriate if a microstructure or final product cannot be manufactured within one fabrication step or if its size does not allow transportation, for example, a big microfluidic device cannot be moved through microchannels (“ship-in-a-bottle”- problem). Single components can then be transported through the channel to de-sired position and can locally be assembled to required size. Lastly, with a technique that also allows disassembling or releasing of components of a microsystem, it can quickly and easily be adapted to new ambient conditions and requirements, without the fabrication of a complete new microfluidic chip.

Different techniques and tools are used to grab, position, and assemble micro-components. Mechanical microgrippers, for instance, provide handling of compo-nents for typical robotic assembling scenarios. Certainly, adhesion forces, for exam-ple, surface tension, electrostatic, or van der Waals forces, overcome gravity forces on the micrometer scale and cause difficulties for releasing free standing components [28]. One promising way to overcome sticking effects of the microgripper is to use techniques without mechanical contact in the first place. Therefore, the use of optical assembling techniques and contactless OTs in particular are promising alternatives.

372 Generation of microstructures with two-photon polymerization

Besides overcoming the difficulties of sticking effects, OTs provide trapping and positioning of objects with a broad range of sizes. Trapping of particles in nanometer scale is supported as well as the provision for particles with diameters of several 10 µm. Due to electronic control and integrated imaging techniques, automatic detec-tion and positioning of multiple trapping spots is feasible. OTs can, therefore, be used for automatic assembling of microcomponents [29]. Furthermore, the high po-sitioning and detection accuracy are beneficial features.

1.4 CONTENTSThe main aim of this chapter is to give an overview of assembling techniques using OTs. More precisely, the focus is on methods that are appropriate for microfluidic applications. Therefore, three main topics are covered:

• First,2PPandthefunctionalprinciplesbehinddifferentphotosensitivematerials are described in detail. Certainly, these fundamentals are important for manufacturing structures on the micrometer scale. Furthermore, several assembling techniques are based on 2PP.

• Second,diverseconnectionandjoiningtechniquesarepresented.Thesearebased on surface chemistry, thermal and photothermal connection, joining by polymerization, and interlocking or form-fit connections. The thematic priority is on the latter technique, which is based on certain microstructures generated by 2PP. The advantages and disadvantages of the listed assembling techniques regarding different application areas are extensively discussed.

• Third,differentapplicationsfortheassembledmicrostructuresarepresented.More precisely, the assembled devices are applied for pumping, mixing, and controlling fluids. As a result, the integration and efficiency of such devices can be approved.

2 GENERATION OF MICROSTRUCTURES WITH TWO-PHOTON POLYMERIZATIONSince recent years, microstructures with feature sizes in the submicrometer play an important role for applications in biology, medicine, micro mechanical systems, and photonics [7,30,31]. The fabrication of microstructures can occur through different techniques based on rapid prototyping. Employed materials have extended, for in-stance, to chemical solutions [32], metal powders [33,34], or polymer powders [35]. However, a greater resolution can be realized with the fabrication of microstructures based on photopolymerization using light as an energy source. Photopolymerization is defined as the conversion of unsaturated liquid molecules to solid macromolecules initiated by photochemical excitation caused by light irradiation. Therefore, photo-thermal processes, which limit the resolution due to thermal energy diffusion, are restricted. Basic components for the photopolymerization processes are monomers

38 CHAPTER 2 Laser-based assembler and microfluidic applications

and oligomers. The solidification of those materials may occur by two reaction pro-cesses, which are polymerization and crosslinking [36–40]. The former is related to the creation of macromolecules caused by a chain reaction, while crosslinking refers to the formation of crosslinks with chemical bonds. The two reaction processes can be distinguished by their quantum yield, which is defined as the ratio of number of bonds formed per absorbed photon. The quantum yield for crosslinking is less than one because each monomer unit requires absorption of at least one photon. In con-trast to photopolymerization via chain reaction the quantum yield can reach several thousands [40]. Thus, a higher number of monomer units can be linked to macromol-ecules simultaneously with the absorption of one photon.

The polymerization process can be divided into three main steps. The first step is called photoinitiation (1). Photoinitiators are included to increase the quantum yield of general monomers and oligomers [36–40]. Furthermore, those molecules increase the initiating efficiency due to a higher sensitivity to light irradiation. They form ini-tiating species of radicals by absorbing suitable photon energy as described in Eq. 2.1

→ →I I R ,hv * * (2.1)

where the photoinitiator is indicated by I, and radical by R*. The symbol I* denotes an intermediate state of the photoinitiator after absorbing a photon.

Afterwards the radicals R* react with monomers or oligomers. The produced monomer radical expands in a chain reaction with the combination of new mono-mers. The chain propagation (2) is described in Eq. 2.2.

+ → → →R M RM RMM RM...Mn

* * * *

(2.2)

The monomer or oligomer unit is here described with M, while Mn is the macro-molecule containing n number of monomer units. The last step of photopolymeriza-tion is called termination (3). Termination means the completion of chain propaga-tion as described in Eq. 2.3 and Eq. 2.4. It is reached when two radicals react with each other.

+ → +RM RM RM Rn m m n* *

(2.3)

+ → +RM RM RM RMn m m n* *

(2.4)

For 3D-lithography processes suitable viscosity of the resin is important to ensure the fabrication process considering the solidification quality of the exposed mate-rial and the solubility of non-exposed areas by the developer. Further major mate-rial properties are high polymerization efficiency upon light irradiation and lower shrinkage after polymerization [41]. For the purpose of photopolymerization, nega-tive resins, which are hardened by the irradiation of light with suitable wavelength, are applied. Commercial products of those photosensitive materials are highly absor-bent in the UV range. Due to a single-photon polymerization process at the surface of the resin using a UV laser, also known as stereolithography, the entire structure

I→hv I*→R*,

R*+M→RM*→MRMM*...→RMn*

RMn*+RMm*→RMm+nR

RMn*+RMm*→RMm+RMn

392 Generation of microstructures with two-photon polymerization

is sequentially created layer-by-layer. The accuracy considering the laser fabrication process could demand submicrometer feature size [42]. But in general, those micro-structures have to be processed afterwards, for instance by etching, to increase the resolution and quality. In comparison to the photopolymerization using single pho-ton, 2PP has some advantages [43–45]. The linear absorption of commercial poly-mers in the near-infrared (NIR) region is negligible. Hence, they are transparent for the wavelength of commonly used ultrashort pulse lasers. Thus, the photopolymer-ization can be initiated within the volume of the polymer. The deflection of the laser focus, for example, by a galvo scanner, enables the fabrication of microstructures with 3D spatial resolution. Fig. 2.1 shows a standard 2PP-setup.

The photopolymerization by two photons is a nonlinear process. 2PP is based on the quasisimultaneous absorption of two photons, most generally referred to 2PA or TPA. The theory of 2PA was first developed by Maria Göppert-Mayer in 1931 [46] and was experimentally observed in 1961 using the then new invented ruby laser [47]. It describes the energy gap excess of an electron in one excitation event by the simultaneous absorption of two photons in a mechanical three-body system [48–50]. A virtual state is formed with the absorption of the first photon. Its lifetime amounts to several femtoseconds. Thus, the second photon has to be absorbed before the vir-tual state decays. High energy density is required to initiate the polymerization pro-cess as 2PA is proportional to the square of intensity [47,51]. This can be achieved by a combination of an ultrashort pulse laser and a strongly focusing microscope

FIGURE 2.1 Standard Setup for Structuring via Two-Photon Polymerization

A femtosecond laser system, for example, a mode-locked Ti:sapphire laser with a repetition rate of 82 MHz, a pulse width of 90 fs and a wavelength of 780 nm, is used in a 2PP-setup. The adjustment of the average laser beam power is realized by a combination of a motorized λ/2-plate and a polarization beam splitter cube. The average power is measured by a photodiode. An acousto-optic modulator is employed as a fast shutter. The laser beam is tightly focused by an oil-immersed objective with a NA of 1.4 into the drop of a photosensitive material. The resist position along the axis can be controlled by mechanical stages. A CCD camera enables the monitoring of the fabrication process. Arbitrary geometries are written by the deviation of the laser beam in the photoresist with the use of a 2D galvo scanner.

40 CHAPTER 2 Laser-based assembler and microfluidic applications

objective. An illustration of the polymerization process with two photons is pre-sented in Fig. 2.2.

Due to the small focal volume, the fabrication of microstructures is time consum-ing. One method to reduce this time is the simultaneous fabrication of the same struc-ture using multiple laser foci [31,52]. Another method is presented by the fabrication of the structure directly in shape [53–55]. For these purposes, the laser beam is mod-ulated with a phase-only SLM. In general, most commercial polymers like urethane acrylates [45,56,57] or inorganic-organic hybrid materials [58,59], which are used for 2PP, are polymerized by the above-mentioned radical reaction. Furthermore, 2PP enables the polymerization of epoxide resins like SU-8 [60–62] as well. The solidification of the material is reached by a photochemical change caused by light irradiation followed by thermal crosslinking. This process is related to the cationic polymerization, which relates to the classification of ionic polymerization.

The voxel or volume pixel is defined as the minimal polymerized structure size in the focal volume of the ultrashort laser pulse. The dimension of the voxel, which is fabricated by one single ultrashort pulse, depends on the threshold behavior of the polymerization. This parameter and also the distance between two voxels are mainly important considering 3D-structuring processes [40,63]. 2PP offers a high resolution of about 100 nm in lateral direction and 500 nm in axial direction using an objective lens with a NA of 1.4 [64].

In general, the minimum resolution of a focused laser beam is achievable by diffraction limit for a microscope described by Ernst Abbe. However, because of the threshold behavior of the nonlinear process, a resolution beyond the diffraction limit can be realized by control of the laser pulse intensity and the number of applied

FIGURE 2.2 Polymerization Within the Volume of a Negative Resin Using 2PP

High energy densities in the focal plane can be obtained applying an ultrashort pulse laser and a high NA objective. The total energy for one excitation event by absorbing two photons is given by the sum of each single photon energy, E = 2hν, where h indicates the Planck constant and ν the frequency of light.

412 Generation of microstructures with two-photon polymerization

pulses [58]. A schematic illustration of the correlation between the voxel size and intensity is shown in Fig. 2.3. The determination of the voxel dimension refers to following assumptions.

First, the lateral light distribution at the maximum focal plane (z = 0) as well as the axial light distribution along the beam axis (r = 0) can be defined by a Gaussian distribution as shown in Eqs. 2.5 and 2.6.

= ⋅−

N r t N t e( , ) ( )r

r0

2 2

02

(2.5)

=+

N z tN t

z

z

( , )( )

1R

02

2

(2.6)

The photon flux is defined by N0(t) = N0. It can be assumed to be constant during one laser pulse due to the requirement of several pulses to reach the polymerization threshold. The Rayleigh length is given by the parameter zR.

Secondly, a negative photosensitive material is polymerized as soon as a certain threshold value of the particle density of radicals ρth is exceeded. The density of radi-cals ρ = ρ(r,z,t) produced by an ultrashort pulse laser can be calculated by

N(r,t)=N0(t)⋅e−2r2r02

N(z,t)=N0(t)1+z2zR2

FIGURE 2.3 Voxel Size as a Function of the Laser Intensity in the Focal Plane

High resolution below the diffraction limit can be obtained for an intensity close above the polymerization threshold based on [65]. Furthermore, optical breakdown characteristics and the suppression of thermal diffusion provide a unique chance to realize subdiffraction-limit fabrication with ultrafast lasers and an intensity threshold above the photoreaction via absorption [45,66].

42 CHAPTER 2 Laser-based assembler and microfluidic applications

ρρ ρ σ

∂∂

= − ⋅x t

tx t N x t

( , )( ( , )) ( , )0 2

2

(2.7)

In Eq. 2.7 the value σ σ η= a2 2 describes the effective cross section of 2PP for the

generation of radicals. The product consists of the ordinary 2PA cross section, σa and the efficiency of the initialization process η < 1 [67]. The primary initiator particle density is defined by ρ0.

An approximation for the voxel diameter dvoxel in the region, where the density of radicals ρ is higher than the certain threshold value of the particle density of radicals ρth is given by

σ τρ

ρ ρ

= ⋅

−

⋅d N t rN n

( , )voxel 0 02 0

2L

0

0 th

InIn

(2.8)

The number of applied pulses is defined by n = νt, where ν is the repetition rate of the laser source and t is the exposure time. The parameter τL denotes the laser pulse duration. According to the approximation of the voxel diameter, the estimation for the voxel length results from Eq. 2.9 considering Eq. 2.6.

σ τρ

ρ ρ

=

−

−l N t zN n

( , ) 2 1Rvoxel 02 0

2L

0

0 th

1/2

In

(2.9)

The ratio of Eqs. 2.9 and 2.8 is > 1 and increases with the increment of laser pow-er or exposure time. To obtain minimum voxel dimensions (lvoxel/dvoxel ≈ 1), which leads and a high resolution, the polymerization of the resin has to occur close to the polymerization threshold [58].

However, sub-100 nm structures already have been fabricated by 2PP or mul-tiphoton polymerization technique. This was realized using modified materials, for example, by adding a cross-linker to a photopolymer [68]. Three-dimensional multiphoton lithography at 520 nm has been used to fabricate polymeric wood-pile photonic crystal structures with nanoscale features of 65 nm by Haske and coworkers [69]. Nanorods with lateral dimensions of 30 nm were produced in the photosensitive material SU-8 by Juodkazis and coworkers [61]. Based on re-polymerization between two structures close to each other, Tan and coworkers [70] have generated even sub-25 nm lines in the resin SCR500. Furthermore, new illumination concepts like the stimulated emission-depletion lithography [71] or focusing the ultrashort laser pulses into the polymer volume without aberration using refractive-diffractive hybrid optics [72] can increase the resolution of the process.

∂ρ (x,t)∂t=(ρ0−ρ (x,t)) σ2N (x,t)2⋅

σ2=σ2aη

dvoxel (N0, t)=r0⋅In σ2N02nτLIn ρ0ρ0−

ρth⋅

lvoxel (N0, t)=2zRσ2N02nτLIn ρ0ρ0

−ρth1/2−1

433 Assembling techniques

Thus, 2PP is a modern tool of maskless microstructure fabrication with high flex-ibility considering the arbitrariness of 3D structures and a high resolution in the submicrometer. Therefore, the process now offers a high potential for microfluid-ics application. Lin and coworkers [73] developed a 3D Archimedes-screw-shaped mixer to disturb a 3D laminar flow direction for enhancing the mixing efficiency. Another method of fluid flow control is given by [10] in an overview, which com-prises the application feasibilities of optical transport, sorting, and characterization of microspheres inside a microfluidic channel. Furthermore, the polymer’s charac-teristics, like elasticity, also enable the sensing of the fluid flow velocity [73] or the fabrication of rotational microjoints based on cross-springs for the development of actuators [74].

3 ASSEMBLING TECHNIQUESFor future nano- and microtechnologies, miniaturization and integration are indis-pensable. Therefore, assembling techniques, which can be defined as bottom-up methods and applied for manufacturing complex microstructures, play an impor-tant role. As described earlier, OTs provide numerous advantages for assembling components in the micrometer range. For instance, optical forces are sufficient for contactless trapping and moving of particles in all three dimensions. By mul-tiplexing the laser beam, several particles or even complex microobjects can be manipulated at the same time. Therefore, these particles can be arranged to certain patterns or structures. In addition, when handling a large number of particles, opti-cal binding forces emerge from multiple scattering, and interparticle interactions occur [26]. Therefore, optical binding offers potential for more advanced self-assembled structures. For faster and automatic arrangement of multiple particles into desired patterns, a closed-loop control can be implemented [75]. Further-more, with integrated imaging recognition automatic sorting of different particles regarding size distribution is feasible. These patterns and structures can be used for photonic applications, for example, where quasicrystalline heterostructures are required [76].

In Fig. 2.4A, a basic experimental design of an OT is illustrated. To facilitate multiplexing, as well as to generate complex beam shapes, a SLM is integrated. Therefore, the beam is first expanded to utilize the active area of the SLM. After modulation and reflection, the beam is projected onto the back aperture of a high NA microscope objective by using two lenses in a 4-f-configuration. The microparticles or microcomponents are typically placed in a small microfluidic chamber, which is covered by transparent cover glasses. For monitoring the trapping and manipulation process as well as for analyzing certain properties of the optical configuration, for example, for determining the trap stiffness, a camera is used. Also, quadrant photo-diodes are typically applied. In Fig. 2.4B–D the phase pattern and related intensity pattern for trapping seven microparticles in a ring-shaped arrangement are shown, respectively.

44 CHAPTER 2 Laser-based assembler and microfluidic applications

However, the imposed assembly requires optical forces and once the laser is switched off, the particles drift apart due to Brownian motion. Thus, they are not durable and further processing is not realizable. To manufacture microsystems which are characterized by a stable connection, joining, or binding techniques have to be applied. The fundamental idea and the repetitive procedures of optical assembling with the aid of joining or connection techniques are:

1. selecting and trapping of a microcomponent or building block;2. transportation and positioning at required position;3. utilizing certain techniques to join or connect the microcomponent with another

or to the substrate; and4. shaping and increasing the size of the required structure by repeating the steps.

In the following paragraphs, the different approaches for joining microcompo-nents are introduced and discussed regarding applicability and microfluidic usage. Furthermore, their advantages and disadvantages are stated.

FIGURE 2.4

(A) Typical configuration of an HOT is illustrated. The phase pattern (B) and related intensity pattern (C) are applied to trap seven microparticles (φ ≈ 5 µm) in a ring-shaped arrangement. The trapping and manipulation process can be monitored using an integrated camera (D).

453 Assembling techniques

3.1 CHEMICAL BONDINGCastelino and coworkers [77] proposed different methods to generate a stable con-nection between spherical particles due to surface chemistry. These chemical ap-proaches are based on gold seeding, complementary DNA linkage, and interaction between two biomolecules, namely streptavidin (SA) and biotin (B). The indirect gold seeding approach involves size enlargement of gold nanoparticles due to gold deposition. Polystyrene (PS) microparticles are coated with gold nanoparticles and positioned next to each other with the use of optical forces. The reaction is started by inducing a seeding agent. The growth of the gold nanoparticles leads to fusion of the coatings and the particles are stably joined. DNA-DNA linkage can be realized by coating microparticles with complementary strands of molecules. Type I and II coated particles are brought into contact and magnesium ions are added to aid hybrid-ization and a stable connection. Similarly, the SA–B bonding can be used. By posi-tioning two B coated particles next to each other and adding SA into the solution, the very strong affinity of the molecules leads to a stable binding force. These methods are sufficient to assemble larger structures with a broad range of different materials, for example, the use of organic and inorganic materials or living and nonliving com-ponents is possible. Therefore, these approaches can also be used for cell marking and tracking in biotechnology.

The biochemical assembly has been experimentally studied with the aid of OTs. Park and coworkers [78] use B coated microparticles based on PS and add these in a SA solution. Instantly, a self-assembling process emerges and chained structures of two or more particles are formed. However, by means of optical forces these build-ing blocks can be trapped and combined by bringing them into contact. SA, which is present in the solution, ensures bonding of the particles. It can be shown, that com-plex planar microstructures can stably be assembled. Furthermore, these structures can be used as microtools for indirect manipulation of other particles, for example, nontransparent objects or biological samples can be moved and positioned.

For all three mentioned chemical approaches, it is necessary to add an initiator after positioning of the particles to trigger the joining process. Ghadiri and cowork-ers [79,80] use SA and B coated microparticles based on PS to avoid this procedure. With the use of OTs, these particles can be trapped and positioned next to each other. By bringing a SA and a B coated particle into contact, the high affinity between the coatings leads to a rigid and stable connection. To distinguish between the different particles, the complementary particles are highlighted by different sizes or optical properties. These varieties can be determined under the microscope, for example, SA coated particles are 12 µm and B coated particles are 3.5 µm in diameter. The alternating structure can fast become larger by attaching further particles and desired shapes and structures can be realized. It can be shown that planar as well as 3D mi-crostructures are achievable [81]. Furthermore, particles based on different materials can be used for this purpose.

Fig. 2.5 shows the assembling strategy for a 3D pyramid composed of micropar-ticles with 9 and 6 µm in diameter. First, substructures are manufactured and suc-cessively stacked onto each other. Also, the combination of different materials is

46 CHAPTER 2 Laser-based assembler and microfluidic applications

achievable. Therefore, desired properties, for example, optical, mechanical, electri-cal, or magnetic properties, can be generated within a hybrid microstructure.

The forces to rupture the SA–B connection can also be determined using OTs [82]. However, this connection is the strongest noncovalent binding known. There-fore, it is widely used in biotechnology, for example, for molecule separation or as a detection system for different targets [83]. Furthermore, particles coated with SA and B are commercially available in numerous sizes and materials (available at, e.g., microParticles GmbH, Germany).

3.2 THERMAL AND PHOTOTHERMAL CONNECTIONLasers are broadly used for cutting, welding, and drilling in different fields of tech-nology on the macroscopic scale. Responsible for melting a substrate is the heat input due to absorption of the laser beam. Additionally, this can be utilized for as-sembling particles at the micrometer range with OTs. High intensities in the order of megaWatt per square centimeter of the tightly focused laser beam are sufficient to heat polymer particles [84].

Won and coworkers [85] deploy absorption of a laser beam to fixate polymer mi-croparticles on a polymer substrate. Thereby, the thermal energy of the laser leads to local melting and the particles can be bonded. To further increase absorption in visi-ble range, Rhodamine-B (Rh-B) is added in the process. The bonding technique is in-vestigated and analyzed regarding various polymer particles and polymer substrates in combination with two different solvents. It can be demonstrated, that arbitrary

FIGURE 2.5 Biochemical Bonding is Applied for 3D Microfabrication

Coated 9 and 6 µm particles are forming substructures, which can be stacked onto each other to manufacture a pyramid [81].

473 Assembling techniques

patterns and structures can be fabricated. Therefore, a particle is first trapped with a focused 1064 nm laser beam and next moved to the required position onto the sub-strate. A second pulse of 532 nm is used to fixate the particle, while the focus is at the interface between the particle and the substrate. The process to fixate one particle occurs within 10 s. The authors present different patterns and can demonstrate that the bonding forces are larger than applied optical forces.

In another experiment, the fixation of polymer microparticles on a glass sub-strate is demonstrated using one wavelength only [86]. Therefore, a PS particle with diameter of 4 µm is trapped and positioned onto a cover glass using a cw laser with a wavelength of 532 nm. Subsequently, the laser power is increased till the particle locally melts. The melting temperature for PS is approximately 240°C. This process can be monitored due to the bright glow of the particle. The focus is then axially lowered towards the cover glass. Once the particle is fused, the laser beam can be turned off. In this experiment a strong bonding force between the particle and the cover glass can be realized. To manufacture arbitrary patterns, multiple particles can be positioned and fixated step by step, as it is shown in Fig. 2.6. The particles can lose their spherical shape and get locally deformed due to high temperatures and melting process.

To manufacture microstructures that are freely movable and not fixated on a sub-strate, the thermal energy has to be introduced in another way. Ghadiri and cowork-ers [87] present an approach to generate a large proportion of thermal energy by heating the surrounding fluid into which particles are added. Therefore, a heater ele-ment is placed around the sample chamber controlled via a thermocouple sensor. In this experiment, poly(methyl methacrylate) (PMMA) microparticles with diameter of 9 µm are used to manufacture microstructures. To reach the melting temperature of PMMA (approximately 105°C), it is necessary to use surrounding liquid with a higher boiling point than water to prevent evaporation. Therefore, ethylene glycol with a boiling point of approximately 197°C was chosen. First, two particles are trapped by generating multiple trapping spots and the fluid in the sample chamber is heated. As the fluid is sufficiently heated up, the particles are joined together and are stably connected.

As it is stated for the macroscopic scale, thermal connections lead to high bonding forces. This can also be demonstrated for applying these assembling techniques for

FIGURE 2.6

(A) Schematic depiction of the thermal bonding process is shown. (B–D) This assembling method can be utilized to generate different patterns of PS particles on the cover glass [86].

48 CHAPTER 2 Laser-based assembler and microfluidic applications

the fabrication of microstructures or micropatterns composed of polymer particles. It is also advantageous that no additives or toxic solvents are required for processing. However, these techniques are restricted only to polymer particles due to relatively low melting temperatures. Up to now, no studies reported the bonding or fixation of glass or metallic particles in liquid due to melting by OTs. In conjunction with biological samples, still, the temperatures used for these assembling techniques would damage biological material or organism in direct contact. Thus, these assem-bling techniques cannot be combined and applied in biotechnology. Furthermore, these temperature-driven techniques require long time periods for assembling larger structures.

3.3 JOINING BY POLYMERIZATIONAs described earlier, 2PP can be utilized to fabricate arbitrary microstructures with high resolution. However, the combination of different materials and multiple func-tions within a microstructure still remains challenging. To take advantage of the as-sembling concept, polymerization can be applied to fixate or join microcomponents composed of different materials and properties.

For photonic applications, for example, photonic crystals, periodically micropat-terns are essential. Mirri and coworkers [88] present an approach to assemble such structures by positioning microparticles and stabilizing them through photopoly-merization. In the process, silica microparticles with diameters of 3.1–7.3 µm are added in a mixture of monomers, crosslinker, and photoinitiator. Additionally, liquid crystals in the solution are used and provide the alignment of the particles to linear chains by self-assembly. Due to the anisotropic media, the particles are guided to obtain these ordered structures. With the use of an OT, these chains can be positioned next to each other to generate a planar surface of tightly packed microparticles. Ir-radiation with UV light over the whole area for 1 min and the chemical reaction is initiated. The polymerization stabilizes the assembled microstructure and a 2D plane is manufactured. Further, the microstructure can be used as a diffraction pattern and is characterized by high temperature stability up to 150°C.

Several groups are studying the polymerization process to join microparticles and to manufacture freely movable arbitrary microstructures [89–91]. Arai and cowork-ers [92], for instance, assemble structures by joining single microparticles step by step due to optically initiated chemical reaction. Furthermore, these structures are not fixated on the cover glass and can further be manipulated with the OT. PMMA and PS particles with diameters in the range of 1–10 µm are dispersed in a sample solution, composed of monomers, crosslinker, and radical photoinitiator. A cw laser beam with a wavelength of 1064 nm for trapping the polymer microparticles is applied. Using galvano scanners or SLM, two particles can be positioned next to each other. To induce photochemical reactions, a second pulsed laser beam with a wavelength of 355 nm irradiates the sample solution between the particles. Polymerization and crosslinking leads to stable connection of the particles. This process of joining two particles takes several seconds. By means of this technique, multiple particles can

493 Assembling techniques

be joined and assembled to desired shapes. The structures are freely movable and can be positioned and rotated by optical forces. Furthermore, they can be applied for indirect movement of biological samples.

Terray and coworkers [93] present a similar technique for photochemical joining of polymer particles. However, this method enables manufacturing of particle chains and more complex structures by using one single wavelength only. A green laser with a wavelength of 532 nm is used to assemble 0.64 and 3 µm polymer particles into linear chains. Therefore, the particles are selectively trapped and joined due to polymerization in the focus of the trapping laser beam. These chains than can be po-sitioned next to each other to assemble complex structures. Furthermore, the combi-nation of different particle sizes within one structure is achievable. These structures can be applied for microfluidics, for example, for valves to control the fluid flow, as will be discussed in Section 4.1.

3.4 INTERLOCKING CONNECTIONExisting joining technologies are usually represented by adhesive bonds resulting in durable and inflexible structures. But joining different subcomponents can also be realized by using interlocking shaped micro-components, which can be released if necessary. Thus, a high surface quality for interlocking parts is inevitable for such assembly procedures with optical tools only. For this purpose, fabrication by 2PP and manipulation by HOTs are applied. Thereby, releasable microparts are fabricated by 2PP for HOT manipulations.

Fabrication by 2PP and assembly by HOTs of interlocking preforms requires the two technical installations. In combination, these two experimental setups are applied to manufacture, manipulate, assemble, and also disassemble complex com-ponents in micrometer range for generating futuristic 3D microrobotic systems. Thereby, desired nonspherical complex microcomponents with or without spherical trapping ports can be generated, developed, and transported into the HOT manipula-tion processing. By applying one of the following assembly technologies, not only durable assembly with spherical structures, but temporary assembly processes by utilizing form-fitting microcomponents become possible.

Spherical ports are often integrated into complex shaped microstructures for simple optical trapping, because trapping characteristics of spheres and thus, as-sembling 3D microstructures with spherical ports, are well known. 3D nonspherical microparts or preassemblies written with 2PP have to be trapped each with multiple spots. Hence, complex shaped 2PP-microstructures need a different handling, for example, specific beam shaped profiles for arbitrary alignment and movement of the microparts, as well as for assembly processes.

Generating complementary microstructures serves the implementation of a re-leasable assembly technology. Within this process, nonspherical microstructures with spherical ports can be manipulated within trapping processes with HOTs. This technique is developed and validated by Glückstad in 2009. Herewith, manu-facturing of specific microstructures with spherical ports is implemented. The use

50 CHAPTER 2 Laser-based assembler and microfluidic applications

of these ports simplifies the handling of the structures and is realized by utilizing counter-propagating beam (CB) profiles, like it is known from the BioPhotonics Workstation [94]. On this occasion, there is no beam modulation required, for example, Bessel or Donut beam profiles, to realize the trapping and assembly. It is to be mentioned, that beam modulation can be necessary for a better manipula-tion and handling of nonspherical shaped microobjects. Glückstad realized a re-configurable microsystem, manufactured with stable mechanical assemblies using optical traps and 2PP-produced structures. Complementary components are linked over dumbbell-like connecting elements as interlocking elements. With this, tem-porary interlocking assembly, as well as disassembly is possible over spherical trapping ports.

Other investigations by Kim and coworkers [95] apply complex 3D microele-ments written directly on the glass surface. This method necessitates a microtip to re-lease the microparts from the surface to receive freely movable microstructures with-in the solvent. This strategy avoids relocation problems of the generated structures in the fluid. The microrings can be moved afterward with multiple HOT traps. The whole assembling process is completed by positioning the microring on a comple-mentary microrod by applying the microtip as fixation tool for this assembly process. A different number of optical traps controls the alignment for the manipulation pro-cess of the microring on the microrod. This is realized by two optical traps, whereas the microgear assembly is implemented by four optical traps on the tiny microrod.

Manipulation of microelements in general requires freely movable microcom-ponents. This is why, after the development process the microelements have to be available freely floating inside the solvent. But the relocation of the microparts is very time-consuming, if the microstructures are not even rinsed out of the manipula-tion area completely. Thus, surrounding compartments shall keep the freely movable microstructures on its defined area for a rapid relocation of the elements inside the solvent. Furthermore, the mechanical microtip, which is applied in Kim and cowork-ers [95], is substituted.

An idea for storage of microparts was developed by Kim and coworkers [96]. This kind of modular system has been implemented containing structures stored as single elements in kind of boxes. The use of multiple optical traps is essential again in this type of storage solution. An idea of placing the manipulative microstructures into separated boxes for group assembly processes is shown in Fig. 2.7.

With a sequential process the fabrication time in the 2PP process with dimensions of 50 × 50 × 30 µm3 takes 2–3 min only. Here, a low laser power of 20 mW but high writing speed of 1.8 m/s can be used for generating inlying high quality structures with a grid as relocation compartment. The inlying complex microelements can success-fully be written with the same writing parameters like the surrounding compartment. Fig. 2.8 illustrates the perforated compartments surrounding the 3D microcomponents. The generated arrays show the results written with different parameters.

Also interlocking 3D puzzle microparts with a size of 5 × 5 µm2 can be as-sembled. Fig. 2.9 gives another application example of geometrical linked subcom-ponents within HOTs.

513 Assembling techniques

FIGURE 2.7 Compartments Filled With Freely Movable Microstructures in HOT Process

FIGURE 2.8 SEM Picture of Perforated Compartments for Inlying 3D Microstructures

FIGURE 2.9 HOT Assembly of 2PP Microsubcomponents

The black crosses indicate the position of the trapping spots. (A) Unassembled freely movable single structures in solvent after transport into the HOT setup. (B) Assembled puzzle structure by utilizing multiple traps simultaneously focused with 100× magnification.

52 CHAPTER 2 Laser-based assembler and microfluidic applications

Horizontal and vertical alignment, as well as rotational movement, are achievable by implementing simple Gaussian beam profiles for the multiple traps. Fig. 2.10 il-lustrates tilting at an angle of 90 degree around z-axis of complex 3D microstructures.

Furthermore, interlocking structures can be trapped and rotated into each other to cause connection [97]. Fig. 2.11 shows how screw and nut shaped microstructures are fabricated by 2PP and joined by HOTs. The microelements are again fabricated inside perforated compartments and are available as freely movable microcompo-nents. The so called optical screw-wrench is realized with screw and nut elements and can be screwed and unscrewed, as required.

Here, it is shown, that releasable microparts are fabricated by 2PP for HOT ma-nipulations utilizing a SLM for generating multiple traps. 3D control of the inlying

FIGURE 2.10

1: CAD file of the generated 3D microcomponents. 2–5: Hole plate with superposed optical traps.

FIGURE 2.11

(A) SEM picture of stable screw connection by OT assembling is shown. (B) The cross-section of the microcomponents is illustrated.

534 Applications for assembled microstructures

microcomponents is possible by applying one to four optical traps for manipulation and assembly procedures with HOTs.

4 APPLICATIONS FOR ASSEMBLED MICROSTRUCTURESFor improving microfluidic systems regarding functionalization, integration, and performance density, the development of next-generation technologies is essential. Presented assembling approaches are promising bottom-up techniques for manufac-turing functionalized microstructures and integrate these in microfluidic environ-ment. In the following, particular assembling techniques are selected and the imple-mentation of these approaches for microfluidic applications is demonstrated.

4.1 OPTICALLY CONTROLLED VALVESAs described earlier, Terray and coworkers [93] use the polymerized joining for manufacturing devices for microfluidic applications (see Section 3.3). More precise-ly, valves are fabricated to control fluid flow in microchannels.

First, a rod-shaped structure composed of seven particles with diameter of 0.64 µm and one 3 µm silica particle is manufactured. Next, the structure is moved into a microchannel with dimensions of 11 µm in width and 3.2 µm in height. The larger particle is used as rotational axis. By applying optical forces, the structure can be held in position, while rotation around the axis is ensured. In Fig. 2.12A–B the mode of operation is demonstrated. The check-valve interrupts the transport of large colloids in one direction and allows the flow of these colloids in the other direction. Thereby, the fluid flow of approximately 0.7 and 2 nL/h, respectively, is causing the valve to swing across the channel.

In Fig. 2.12C–D the manufactured valves are applied for opening and closing certain microchannels. Therefore, the microstructure is moved into a “T”-shaped microchannel. The valve is deflected by optical forces to allow flow either through the top or the bottom microchannel.

FIGURE 2.12 Polymerized Joining is Used to Manufacture Microvalves and to Control Fluid Flow Through Microchannels

Based on schematic drawing on Terray et al. Fabrication of linear colloidal structures for microfluidic

applications. Appl Phys Lett 2002;81:1555 [93].

54 CHAPTER 2 Laser-based assembler and microfluidic applications

4.2 MAGNETIC MICROROTOR: FLOW FIELD DETERMINATION AND PUMPINGAs described earlier, pumping and mixing of small volumes of fluids are basic pro-cesses for microfluidic applications. In the following paragraphs, optical assembling and characterization of a microrotor for these purposes is presented. Furthermore, the rotor is integrated in a microchannel with specific geometry to generate a directed fluid flow, as it is required for pumping applications on a microfluidic chip [98]. Therefore, the biochemical connection (see Section 3.1) is utilized to assemble mag-netic particles to desired rotor shapes. These hybrid magnetic rotors can be actuated by an outer rotational magnetic field and the surrounding fluid flow can be optically analyzed using a probe particle.

Although rotation of microstructures with optical forces is generally possible, the actuation due to a rotational magnetic field contains some advantages: Elec-tronic components can be integrated on a small chip and provide very precise and locally controllable magnetic fields [99]. OTs in comparison are difficult to imple-ment on a small chip. Typically these systems require a microscope, several optical components and a laser system. Furthermore, magnetic forces are relatively high and can, therefore, induce high rotational speeds.

4.2.1 Assembling magnetic rotors with different shapesTo actuate the microrotor with magnetic forces, here, magnetic microparticles are the crucial components. More precisely, magnetic nontransparent silica particles are assembled together with transparent silica particles. To join the different particles, the biochemical connection is selected, because it is beneficial to apply a stable con-nection for the purpose as a microrotor. On the other side, it is not required to have a releasable connection, as it would be possible with interlocking or form-fitting connections. Therefore, the particles are coated either with B or SA and dispersed in an aqueous solution. Due to the high concentration of the magnetite inside the magnetic particles, these particles cannot be trapped with the OT. The laser light is absorbed and the particles can be destroyed due to heat input. To ensure assembling of the particles, a B coated transparent particle is trapped and moved to a SA coated magnetic particle. Due to the high affinity of the biomolecules, a stable connection is succeeded. The particle composition is then moved via the transparent particle to another magnetic particle and a rod-shaped structure is formed. Here, the biochemi-cal connection enables the combination of two different materials composed in one microstructure. Whereas these components are appropriate for different functions: The magnetic components are utilized for magnetic actuation and rotation; the trans-parent particle is used for trapping and positioning the rotor and holding it at its rotational axis during rotation.

With this “pick-and-place” approach, different rotor geometries can be fabricat-ed, as presented in Fig. 2.13. The examples indicate that rotors with different sizes (three- or five-particle rotors) can be manufactured and also the shape (S-shaped rotor) can individually be adjusted [100]. The dark and bright circles represent the

554 Applications for assembled microstructures

magnetic and transparent particles, respectively. Utilizing this technique, a flexible design of magnetic microrotors with desired properties regarding the flow field can be achieved.

Rotation can be induced by an external magnetic field (Fig. 2.14). Four electro-magnetic coils are arranged around the sample chamber and get addressed through a stepper motor driver. As the current at the coils is successively applied, the rotor follows the magnetic field. The maximum rotational speed of the rotor is restricted by the size and contributed flow resistance, the concentration of magnetic material, as well as the magnetic field strength.

For the five-particle rod shaped rotor a rotational speed of 390 rpm could be achieved.

FIGURE 2.13 The Chemical Bonding Enables the Manufacturing of Magnetic Microrotors With Different Sizes and Shapes

The light and dark spheres indicate the transparent and magnetic particles, respectively. The particles are approximately 2.7 µm in diameter.

FIGURE 2.14 With Rotation of the External Magnetic Field (B), the Microrotor Follows as Indicated

A maximum rotational speed of 390 rpm could be achieved.

56 CHAPTER 2 Laser-based assembler and microfluidic applications

4.2.2 Measuring the flow fieldThe flow field of the rotors can theoretically be described and experimentally be investigated by means of the HOT [101]. By generating a second trapping spot, a probe particle can be positioned in the proximity of the rotor. With rotation of the microstructure, a surrounding fluid flow is induced. The frictional force displaces the probe particle from its equilibrium position of the optical trap and by measuring this displacement the fluid velocity at the certain position can be calculated. Azimuthal as well as radial flow components can be determined and the findings agree to theoreti-cal predictions.

The azimuthal velocity linearly increases with the rotational frequency of the rotor. A marked difference can be found regarding the length of the rotor. The rod shaped five-particle rotor generates higher fluid flow velocities as the three-particle rotor. Furthermore, the fluid velocity distinctly decreases with increasing the dis-tance to the rotor. Evaluation of the radial flow component reveals that there is an oscillating fluid flow with twice the rotor frequency. This could not be demonstrat-ed for microfluidic systems rotating about their center in an axisymmetric manner [102,103]. Thereby, no radial velocity component has been detected.

4.2.3 Directed fluid flowFinally, the assembled microrotor can be implemented into an appropriate micro-channel to generate a directed fluid flow, as it is required for pumping applications [98]. The microchannel is produced by means of 2PP technique. The design shows a rectangular cross-section area of approximately 76 µm2. A semicircular protrusion is applied in the middle of the microchannel. This symmetry-breaking design promotes the generation of directed fluid flow. After moving the magnetic rotor to required position, the outer magnetic field can be activated to start rotation. By placing a small tracer particle inside the channel, the emerged fluid velocity can be measured. At a rotational speed of 120 rpm, the analysis of the movement of the tracer particle shows an average velocity of approximately 450 nm/s (Fig. 2.15).

It could be demonstrated, that the manufacturing of hybrid magnetic microrotors by means of the biochemical assembling technique is realizable and can sufficiently

FIGURE 2.15

(A) Schematic depiction of the implemented microrotor in a microchannel is shown. (B) The assembled rotor generates a directed fluid flow, as it is indicated by a tracer particle. The dots represent the position of the tracer particle in time steps of 4 s [98].

574 Applications for assembled microstructures

be applied for a typical microfluidic application. Therefore, easy handling and flexible design of the microrotors are feasible. The fluid velocity in the microchannel shows efficient pumping of low amount of liquids.

4.3 MICROROTOR ASSEMBLY USING SCREW CONNECTIONIt has been demonstrated, that the screw connection can be applied to fixate and re-lease microcomponents. In the following, the screw connection is used to assemble a microrotor, which can be actuated for pumping or intermixing fluids on a microflu-idic chip [104]. The screw connection is applied to hold a microrotor on its rotational axis. All necessary components are fabricated using 2PP.

In Fig. 2.16 the assembling process is illustrated. The rotational axis, which is composed of a cylinder having a thread on top, is fixated on a cover glass. The freely movable rotor has four blades to displace surrounding fluid and is approximately 26 µm in diameter. It can be trapped and positioned utilizing optical forces. To ensure ease of handling, the rotor has four spherical ports. Therefore, it can be moved (A) and assembled with the rotational axis (B). The nut is also featured by four spherical

FIGURE 2.16 Assembling Process of the Microrotor is Illustrated

(A) The rotor is moved and positioned over the rotational axis; (B) the rotor is assembled with the axis; (C) the nut is positioned over the axis; (D) the nut is screwed to fixate the rotor.

58 CHAPTER 2 Laser-based assembler and microfluidic applications

ports and can be positioned (C) and screwed together with the rotational axis (D). Therefore, the screw connection ensures fixation of the rotor on its rotational axis. In Fig. 2.17, an SEM image of the assembled microfluidic device is presented.

In addition, the rotor can be actuated in liquid environment using optical forces. Due to rotation of the four trapping spots by the use of a SLM, the microrotor fol-lows, and a rotational speed of approximately 25 rpm can be achieved in both rotat-ing directions. Therefore, it can be used for mixing or pumping applications in a microfluidic channel. Furthermore, due to the screw connection and related capabil-ity of disassembling, rotors can be exchanged and required flow properties can be adjusted without the fabrication of a complete new microfluidic chip.

5 CONCLUSION AND OUTLOOKIn this chapter, it is shown, how laser-based systems can be applied for manufacturing, manipulation and assembly processes for microfluidic applications. Here, opti-cal tools are applied for precise fabrication within the laser direct writing process 2PP. For 3D control by HOT of microstructures specific connection techniques are implemented, like biochemical, thermal and photothermal linkage, polymerized joining, but with main focus on interlocking techniques for complex microcompo-nents. Hence, releasable joining of microcomponents and thus temporary assembly of microelements can be accomplished. The assembly process of such complex mi-crosystems necessitates not only precise vertical and horizontal movement, but also rotational alignment of the fabricated microcomponents by HOT.

Selected application examples are described in detail and demonstrate how mi-crofluidic systems are improved regarding their functionalization and integration into, for example, 2PP generated microchannels. Assembly processes for magnetic microrotors are given as a result of combining magnetic nanoparticles with silica

FIGURE 2.17 SEM Image of the Assembled Microrotor

The rotor is fixated on its rotational axis and can be actuated using optical forces.

59References

microspheres. Thus, microstructures can get magnetic functions and are therefore able to interact with outer rotational magnetic fields. Assembly of functionalized microspheres is done with “pick and place” technique. With this, a surrounding fluid flow can be analyzed using a probe particle. For a five-particle rod shaped rotor, ro-tational speed of 390 rpm could be achieved. Additionally, the flow field and direct fluid flow can theoretically and experimentally be described and investigated, result-ing in the fact, that fluid velocity decreases with increasing distance from the rotor, like it is shown in the comparison of a five- and three-particle rotor. Higher fluid ve-locities could be detected with the five-particle rotor. Latest researches have shown that rotational speed of 120 rpm can be achieved by integration of the assembled microrotors into 2PP microchannels, resulting in a directed fluid flow.

Combination of both techniques—2PP and HOT—opens up a completely new field for rapid microprototyping, assembly and, thus, generation of complex microflu-idic devices as well as microrobotic systems. Worth special mention is the introduced assembling method, which can also be applied for generating hybrid multi-functional assemblies by combining different materials. This is still a challenging task for future applications. Based on these researches diode-based femtosecond lasers can be ap-plied in futuristic applications, resulting in an economical miniaturized setup with for functionalized arbitrary microstructures. SLM-based simultaneous generation of multiple freely movable microstructures could be possible as well, by integrating an SLM into a miniaturized experimental setup. Further techniques are still required as well as microfluidic applications still need to be expanded.

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65

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00003-3Copyright © 2017 Elsevier Ltd. All rights reserved.

David Phillips*, Stephen Simpson**, Simon Hanna†

*University of Glasgow, United Kingdom; **Institute of Scientific Instruments of the CAS, Brno,

Czech Republic; †University of Bristol, Bristol, United Kingdom

CHAPTER OUTLINE

1 Introduction and Background ............................................................................... 652 Theory ................................................................................................................ 67

2.1 Introduction ....................................................................................672.2 Shape-Induced Optical Forces ...........................................................672.3 Calibration of Traps Containing Nonspherical Particles.........................76

3 Experimental Realizations ................................................................................... 803.1 Microtool Fabrication .......................................................................803.2 3D Tracking .....................................................................................853.3 3D Optical Control ...........................................................................86

4 Applications ....................................................................................................... 885 Conclusions and Future Prospects ........................................................................ 93References ................................................................................................................ 94

1 INTRODUCTION AND BACKGROUNDThe vast majority of experiments with optical tweezers have employed microspheres to act as force transducers [1,2], image surfaces [3,4], and extract a wealth of addi-tional information about their local microscale environment [5,6]. Microspheres are easy to track (crucial as all of the above is revealed by quantitatively observing their motion [7,8]), and readily available in large numbers and in a variety of sizes and materials. In particular, force transduction using optically trapped microspheres has enjoyed great success: the application and measurement of picoNewton scale forces has uncovered an abundance of micromechanical detail of the operation of biologi-cal molecular motors [9,10]. However, increasing the complexity of a microprobe’s shape opens up a range of new experiments previously inaccessible with the use of microspheres alone [11,12]. This chapter details some of the work investigating the deployment of optomechanical microtools: free-floating nonspherical microscale

Optomechanical microtools and shape-induced forces

3

66 CHAPTER 3 Optomechanical microtools and shape-induced forces

structures that can be controllably manipulated and “brought to life” using optical forces, while their motion is tracked in exquisite detail to make a range of new types of quantitative measurements on their surroundings. An example of such a device is shown in Fig. 3.1 [13].

But what are the limitations of the humble microsphere? The optical trapping of microspheres means they are necessarily immersed in the high intensities of a focused laser beam. Therefore they cannot be used to probe samples that may be sensitive to these intense optical fields. Measurements using microspheres can also be adversely affected by highly scattering samples that disrupt the trapping beam, potentially altering the trapping potential in an unknown way, and also affecting the accuracy of optical position tracking. In addition, while the translational motion of microspheres can be constrained and controlled by a laser beam, optically trapped homogeneous microspheres still undergo free rotational diffusion—these rotational degrees of freedom cannot easily be controlled or measured. Therefore the contact point of the microsphere and a sample can never be fully controlled. Finally, the minimum contact area of a microsphere probe with a sample is related to its radius, the minimum size of which is typically on the order of a few hundred nanometers. Reducing the size of the microsphere beyond this limit makes it more challenging to stably trap and track, and increases the level of residual Brownian motion [14].

The design of the optomechanical microtools, such as shown in Fig. 3.1 and de-scribed in this chapter, aims to overcome some of these limitations. Initial work focused on fabricating microtools that possess spherical handles, so that they inherit useful stable trapping properties of microspheres, but also have a sharp tip, physically separated from the trapping handles, that can be used to interact with a sample. Using three handles with this architecture enables control over all six degrees of freedom of the microtool—three dimensions of translational and rotational motion [15]. As the trapping behavior is primarily governed by the handles, such a device may possess an arbitrarily sharp tip,

FIGURE 3.1 An Example of a Microtool

(A) Schematic representation. (B) Optical micrograph.Adapted from Phillips DB, et al. An optically actuated surface scanning probe.

Opt Exp 2012;20: 29679 [13].

672 Theory

now limited by fabrication constraints and not trapping or tracking properties. Further work has moved beyond spherical handles and the technical details outlined earlier, and begun to more widely explore new opportunities brought by shaping particles more gen-erally in optical fields [16]. We also note that over the last decade, a number of different groups have worked on this technology [17–20], and a range of different approaches are detailed in the other chapters (Chapters 4, 6, 7, 9, 12).

This chapter is separated into two parts. The first part details the underpinning theory describing how complex shapes behave in optical fields. This part encom-passes a review of the theory, which leads to the concept of shape-induced forces, enabling the optical forces felt by a structure to be tuned by modifying its shape in a general way. Next follows the theory underpinning the rigorous calibration of op-tomechanical microtools, crucial to transforming them from floating structures into quantitative devices. The second part describes experimental implementations of op-tomechanical microtools. Their fabrication, 3D tracking and control are reviewed, followed by applications including the development of a new form of all optical scanning probe microscope [16]. The flexibility provided by the ability to design ar-bitrarily shaped structures makes possible entirely new experiments and techniques, and at the end of the chapter we look to the future of this emerging field.

2 THEORY2.1 INTRODUCTIONAs indicated earlier, the behavior of dielectric spheres in optical tweezers has been widely studied and is well understood. However, once the symmetry of the trapped particle is reduced, a range of new trapping possibilities occur which have been re-viewed elsewhere [12,21], with the lowest symmetries allowing full control over the 3D position and orientation of the particle. It is helpful to review the underly-ing principles and trends that are observed, in order to gain an understanding of the expected trapping behavior from a particular class of shape. Optical anisotropy and variations in composition further enhance the degree of control that may be possible, as would different types of trapping beams. However, here we will restrict our atten-tion to homogeneous, nonabsorbing, optically isotropic particles trapped in a linearly polarized Gaussian beam.

2.2 SHAPE-INDUCED OPTICAL FORCES2.2.1 The Rayleigh regimeThe forces acting on a sphere are usually expressed as a combination of a scattering force and an intensity gradient force. The behavior of the trapped sphere results from a balance between these forces, and will depend on its radius (r) compared with the wavelength of the trapping laser (λ) and its refractive index relative to that of the me-dium. For small particles with r ≪ λ, the particles are treated as point dipoles, and the optical forces are given by the Rayleigh equations [22]:

68 CHAPTER 3 Optomechanical microtools and shape-induced forces

πλ

∇

=−+

=− −

+

FI

c

r N

Nn

Fn r N

NE

128

3

1

2

2

1

2

scatter0

5 6

4

2

2

2

m

gradientm3 3 2

22

(3.1)

where N = np/nm, with np being the refractive index of the particle and nm that of the medium. I0 is the intensity of the trapping beam and ∇E2 is the local gradient of the square of the electric field. Generally speaking, the sphere will be trapped a little “downstream” of the focus of the optical tweezers, with the precise position varying with r and N.

While the Rayleigh approximation is helpful in acquiring a qualitative under-standing of the trapping behavior of spheres, most trapping experiments are per-formed with larger spheres, with r 1 µm, where the Rayleigh approximation does not apply. This is known as the Mie regime [23] and to obtain a reliable estimate of the optical forces involved, some form of computational approach must be employed.

2.2.2 Force and torque calculation in the Mie regimeA number of computational methods are available for calculating optical forces in the Mie size scale (r ≈ λ). The three techniques that are most commonly used are known as the T-matrix method, the finite-difference time-domain (FDTD) approach and the discrete dipole approximation (DDA). All share the advantage that a degree of shape anisotropy may be included in the calculation. In each case, repeated calcu-lations of forces and torques for varying position and orientation are used to identify the equilibrium trapping coordinates of each particle. In contrast to the Rayleigh equations [Eq. (3.1)], these computational approaches yield a single optical force, that is, gradient and scattering forces are not separated.

In the T-matrix approach, the incident, scattered, and internal fields are expressed as sums of vector spherical wave functions (VSWF) [24–29]. Appropriate VSWF coefficients are available, for example, to describe an incident Gaussian beam. For a given particle, the T-matrix links the incident and scattered sets of coefficients. Calculation of the optical forces and torques follows by integrating the cycle-average Maxwell stress tensor, 〈TM〉, over a surface enclosing the particle:

∫∫ΓΓ

= ⋅

= − ⋅ ×

d

d ˆs

s

M

Mr

F T S

S T r (3.2)

T-matrices are readily available for spheres and ellipsoids but, for lower symme-try particles, some computational effort is required to obtain them. However, once the T-matrix is obtained, calculations of forces and torques may proceed rapidly. A popular implementation of the T-matrix method, the “optical tweezers computational toolbox” is available from the Queensland group [26].

Fscatter=I0c128π5r63λ4N2−1N2+22nmF

gradient=−nm3r32N2−1N2+2∇E2

F=∫sTM⋅dSΓ=−∫srdS⋅TM×rˆ

692 Theory

The FDTD method is conceptually simpler than the T-matrix approach [30]. The trapped particle and trapping beam are represented on a lattice, with Max-well’s curl equations being integrated over time until the required convergence is obtained. Forces and torques are calculated as earlier, using the Maxwell stress ten-sor [Eq. (3.2)]. Typically, the lattice parameter needs to be ∼ λ/20 or less in order to achieve sufficient accuracy [31], which means that force calculations in conventional optical tweezers can be computationally expensive due to the volumes represented by the lattice. However, this method is ideally suited to the comparatively small vol-umes involved in near-field nanoplasmonic traps [32].

The DDA method was originally developed to calculate scattering from interstel-lar dust [33,34]. The approach combines the speed of the T-matrix method with the flexibility of FDTD. The trapped particle is represented as a 3D array of point po-larizabilities, interacting with the external field and with each other. Self-consistent solutions are obtained for the polarizabilities; forces and torques follow by summing contributions from each point in the array [34–36]. As with the FDTD method, the spacing must be chosen to allow resolution of both the particle shape and the incident optical field [37,38].

2.2.3 Equilibrium trapping of nonspherical particlesThe simplest distortion of the sphere is to stretch or compress it along one axis, pro-ducing a uniaxial spheroid. T-matrix calculations [28] indicate that the preferred ori-entation of prolate (cigar-shaped) spheroids depends on whether the particle is long or short compared with the beam width. For a long prolate spheroid, the preferred orientation is parallel to the beam axis (Fig. 3.2A) whereas short prolate spheroids will preferentially orient perpendicular to the trapping beam and parallel to the polar-ization direction (Fig. 3.2B). For oblate (flattened) spheroids (Fig. 3.2C) the particle

FIGURE 3.2

Schematic illustration of the typical trapping configuration of (A) long prolate spheroids, (B) short prolate spheroids, and (C) oblate spheroids. The trapping beam travels parallel to the vertical axis and the polarization direction is taken as parallel to the near-horizontal axis shown in the figure.

70 CHAPTER 3 Optomechanical microtools and shape-induced forces

will tend to lie in the plane defined by the beam axis and polarization direction, with its short axis parallel to both. The principle operating here is that the dielectric particle should achieve maximum overlap with the most intense parts of the trapping beam. This may be expressed physically by saying that we are minimizing the elec-tromagnetic energy functional:

∫ ∫ ∈ω

=c

( )1

2

12

2

Edr

drHH

Df (3.3)

which is achieved by maximizing the overlap between the electric field intensity and the permittivity distribution.

A similar behavior is expected for nanowires and cylinders [38–40]. A single long nanowire will tend to trap vertically (Fig. 3.3A). However, a wider cylinder will trap with an oblique orientation (Fig. 3.3B) because the longest axis is actually the diagonal. The effect will become more pronounced as the cylinder becomes broader.

Distorting the sphere along a second axis produces a general biaxial ellipsoid. DDA calculations indicate a generic type of behavior [41]. Where the three axes of

Ef(H)=12∫drH2∫dr1∈wcD2

FIGURE 3.3

Schematic illustration of the typical trapping configuration of (A) long nanowires, (B) cylinders, and (C) general ellipsoids. The trapping beam travels parallel to the vertical axis and is polarized parallel to the near-horizontal axis shown in the figure. (D) Illustration of the horizontal trapping of a cylinder using two laser traps.

712 Theory

the ellipsoid are distinctly different lengths, the tendency will be for the longest axis to align with the beam axis, the second longest axis to align with the polarization direction, and the shortest axis to be perpendicular to both (Fig. 3.3C).

A consequence of the overlap criterion [Eq. (3.3)], is that it will not always be possible to trap a particle with the required orientation, using a single trap. For ex-ample, if it is required to trap a long nanowire horizontally, for example, so that it may be used to apply a lateral force to a biological cell or some other sample, two or more traps will be required as indicated in Fig. 3.3D. The use of multiple traps will be discussed further, below, in the context of trapping extended structures.

In some of the examples shown in Figs. 3.2 and 3.3, the idea of a polarization torque is invoked, which tends to lead to one of the larger particle dimensions to align with the polarization direction when the beam geometry permits. A T-matrix and FDTD study of the polarization torque, has been used to determine the orienting behavior of a particle of general cross-section [42]. The torque acting on a general particle about the beam axis (z-axis), can be expressed as:

Γ α α β= +( ) sin (2 )A+ Bz (3.4)

where A, B, and β are constants which depend on the rotational symmetry of the particle, and α is the angle taken about the beam axis, measured from the polariza-tion direction. β is dependent on the choice of initial orientation of the particle, and hence its value may vary. However, the values of A and B are closely related to the point-symmetry of the particle. There are two symmetry classes to consider: Cn with n-fold rotational symmetry about the beam axis and Dn which possess both n-fold rotational symmetry and a set of mirror planes parallel to the beam axis. The shapes are illustrated in Fig. 3.4.

Γz(α)=A+B sin (2α+β)

FIGURE 3.4 Cross-Sections for Particles With Different Rotational Symmetries

In order to ensure the cross-section shown is held perpendicular to the laser beam, the particle itself may be extended parallel to the beam axis, or be fabricated with a shaft that will trap parallel to the beam axis.

72 CHAPTER 3 Optomechanical microtools and shape-induced forces

As might be expected, a particle with point-symmetry D2 shows twofold rotational symmetry in its trapping orientations, with respect to rotations about the beam axis. This corresponds to A = 0, B ≠ 0 in [Eq. (3.4)], with the preferred orientations corre-sponding to the longer axis parallel to the polarization direction. There will be mirror planes in the particle which will be parallel and perpendicular to the polarization di-rection. The orientation is similar for D1. Again we observe A = 0, B ≠ 0, but this time the single mirror plane can be parallel or perpendicular to the polarization direction, provided that the longest axis in the trapping plane (x-y plane) remains parallel to the polarization. For Dn, with n ≥ 3, A = B = 0, and Γz = 0, that is, there is no preferred setting angle about the beam axis. The natural extension of this would be to n = ∞, corresponding to the symmetry of a cylinder.

For the chiral systems, Cn, a very different behavior is observed. For Cn, with n ≥ 3, B = 0, and Γz = A, there is a constant torque and hence a constant rotation about the beam axis. For C1 and C2, A ≠ B ≠ 0; the precise details of the particle shape will determine whether continuous rotation is observed, or whether the polariza-tion effect dominates. The general principles for equilibrium trapping orientations of nonspherical particles are summarized in Box 3.1. Chapter 4 further explores the design of structures for light-driven rotations.

2.2.4 Nonequilibrium optical forcesIn the previous section, equilibrium trapping was considered, in which the optical forces acting on each particle were balanced. In most cases, the optical torques were

BOX 3.1 EQUILIBRIUM TRAPPING COORDINATES OF NONSPHERICAL, ISOTROPIC DIELECTRIC PARTICLES• Thepositionandorientationofanon-sphericalparticleinaGaussiantrapwillbeabalance

between the need to maximize the overlap of the particle’s permittivity with the most intense parts of the beam, and the radiation pressure.

• Forparticleswithatleastonedimensionthatislargecomparedwiththebeam-waist,thelongestaxis of the particle will align with, or close to, the beam axis (z axis), while the second longest axis will align with the polarization.

• Shorterparticleswilllieinaplaneperpendiculartothebeam-axis(x-y plane), with longest axis parallel to the polarization.

• Forparticlesalignedwiththebeam-axis,thesettinganglewithrespecttothepolarizationdirection depends on the cross-sectional symmetry:- Prolate uniaxial symmetry, D

∞, and rotational symmetry, Dn, with n ≥ 3: no preferred

orientation;- General ellipsoids, including oblate spheroids, with D2 symmetry in cross section: the

longest axis in the x-y plane will align parallel to the polarization;- Particles with a single mirror plane parallel to the beam axis, but no rotational symmetry

(D1): the longest axis in the x-y plane will align parallel to the polarization direction, and the mirror plane will be either parallel or perpendicular to the polarization;

- Chiral symmetry, Cn, with n ≥ 3: no preferred orientation but continuous rotation;- Chiral symmetry, C1 and C2: no specific orientational preference, but may show continuous

rotation depending on details of the particle’s shape.

732 Theory

also balanced, the exception being particles with chiral symmetry (Cn) which could at least be regarded as in a steady-state. However, when a particle is used to apply or measure a force or torque, it is inevitably displaced from its equilibrium coordinates; the extent to which it moves depends on the trap stiffness which, in turn, depends on properties of both the trapping beam and the particle. In order to develop a deeper understanding of the factors that govern trap stiffness, it is helpful to introduce the concept of the optical force and torque densities.

The idea of the force (or torque) density is that each volume element of a scatter-ing particle will contribute to the total force (or torque) that is acting. The total force (or torque) is obtained by integrating the appropriate density over the volume of the particle. There are several ways of expressing the force and torque densities, which differ in detail, and give slightly different values point-to-point within the particle. This issue has been the subject of considerable discussion [43–49]. However, despite these differences in detail, the integrated forces and torques are generally equiva-lent. In this review, we will consider the force and torque densities due to Brevik [50], which provide a convenient and easy-to- calculate framework within which to explain the phenomenology of trapping.

According to Brevik, the cycle-averaged optical force and torque densities may be written as [50]:

∆ ∈δ= ℜ × + −

= × + ℜ × + ℜ ×

1

2( )

1

4( ) ˆ

1

2( )

1

2( )

* 2surface

* *

f B j E r r n

t r f D E B H

(3.5)

where B is the magnetic induction, j is the current density, ∈∆ is the change in permittivity on crossing the surface at rsurface, H is the magnetic field strength, D is the electric displacement and E is the electric field, and n is the outward normal at the particle surface. In the absence of dielectric or magnetic anisotropy, or optical absorption, these expressions reduce to:

∆ ∈δ= −

= ×

1

4( ) ˆ2

surfacef E r r n

t r f (3.6)

It is clear from Eq. (3.6), due to the presence of the Dirac delta function, that the force density is only nonzero on the surface of the particle, and then it is di-rected parallel to the outward surface normal (assuming the normal trapping case with np ≥ nm). ∆ ∈ is the difference in dielectric permittivity between the particle and the medium, so it is apparent that the force density, and ultimately the total force, is maximized when a surface with a large step-change in refractive index is placed in the highest intensity part of the trapping beam.

The operation of the force and torque densities is illustrated in Fig. 3.5, and summarized in Box 3.2. Fig. 3.5A shows a sphere placed on the axis of an opti-cal trap, some distance from the stable trapping position. From Eq. (3.6), the only contributions to the force density are on the sphere surface and, due to the

f=12ℜ (B×j*)+14 E2 ∆∈δ (r−rsurface) -nˆt=r×f+ 12ℜ (D×E*)+12ℜ (B×H*)

∆∈

nˆ

f=14 E2 ∆∈δ (r−rsurface) nˆ -t=r×f

∆∈

74 CHAPTER 3 Optomechanical microtools and shape-induced forces

spherical symmetry, all off axis contributions will cancel out, leaving an axial force directed toward the trapping position and no resultant torque. If the sphere is displaced laterally, the surface on one side will move into a more intense re-gion and, on the other, into a less intense region (Fig. 3.5B) resulting in a net restoring force (and still no resultant torque). Fig. 3.5C shows a similar situa-tion with an ellipsoid rotated away from equilibrium; the origin of the restoring torque is apparent.

A corollary to the optical force density description is that it is possible to use this idea to optimize optical traps for particular applications. For example, in Fig. 3.3D, a cylinder is shown trapped horizontally by two Gaussian beams. This is reproduced in Fig. 3.6A, which also shows the force density. Since virtually all the force density contributions are perpendicular to the curved surface of the cyl-inder, the lateral trap stiffness will be negligible parallel to the axis of the cylinder. However, by moving the traps toward the ends of the cylinder (Fig. 3.6B), nonzero contributions will arise from the flat ends, allowing the lateral trap stiffness to be tuned [39,51].

BOX 3.2 THE OPTICAL FORCE AND TORQUE DENSITIES FOR NONABSORBING, ISOTROPIC, DIELECTRIC PARTICLES• Theopticalforcedensityinanonabsorbingdielectricparticleisnonzeroonlyatthesurfaceof

the particle, where it acts parallel to the outward surface normal at each point. Thus, only the surface contributes to the optical forces and torques.

• Theopticalforcedensityisproportionaltothechangeindielectricpermittivity,andthebeamintensity, at the surface of the particle.

• Thetorquedensityofanopticallyisotropicspheredependsonlyonthedistributionofforcedensity at the surface.

FIGURE 3.5 Illustration of the Concept of Force Density for Particles in Optical Tweezers

(A) A sphere on the axis of the beam has a symmetrical distribution of force density and will experience at most an axial force. (B) The sphere in (A) is displaced to one side, and force contributions are observed which push the sphere back toward the axis. (C) A spheroidal particle has an asymmetric force density which generates a restoring couple.

752 Theory

2.2.5 Nonconservative forces in optical tweezersA further application of the force density description is to the design of optically controlled probes utilizing nonconservative forces. Although optical tweezers are in-herently nonconservative, in that the forces result from a flow of optical momentum through the system, it is usual to regard the trapped particle as moving in an effective harmonic potential, with restoring forces and torques to bring it back to equilibrium. However, by suitably shaping the particle, it is possible to generate forces which are not restoring [16,51]. An example is shown in Fig. 3.7. In this case, a probe consist-ing of conical sections is supported in two optical traps. Force density considerations suggest that there will be a net force to the right in the figure, at least until the ends of the cones reach the trap. This arrangement forms the basis of the constant force probe illustrated in Figs. 3.19 and 3.20.

FIGURE 3.6

(A) A cylinder held horizontally in two traps, where the traps are positioned away from the ends, will have a negligible horizontal axial trap stiffness. (B) When the traps are moved toward the ends of the cylinder, there will be additional contributions to the force density from the end faces.

FIGURE 3.7 Schematic of a Composite Particle Consisting of Two Conical Sections Joined by a Cylinder

The conical sections are placed in two traps; the force density has uniaxial symmetry about the cone axis, but there is a net force acting to the right in the figure, which will remain constant provided the conical surfaces remain in the traps.

76 CHAPTER 3 Optomechanical microtools and shape-induced forces

Other applications of lateral forces through particle shaping include the optical wing, which consists of a semi-cylinder which moves with a component of velocity transverse to the applied optical field [52,53].

2.3 CALIBRATION OF TRAPS CONTAINING NONSPHERICAL PARTICLES2.3.1 Trap stiffnessThe calibration of optical tweezers is crucial if they are to be used to apply or measure forces. Forces may be applied to a sample by moving the beam so that the trapped particle interacts with the sample in some way; the value of the force is related to the displacement of the particle from its equilibrium trapping position by:

= − ⋅ −( )eqmF K q q (3.7)

Here, we use F to represent a generalized force (including torques), that is, F = (Fx, Fy, Fz, τx, τy, τz), and q to represent a generalized coordinate (including angles), that is, q = (qx, qy, qz, θx, θy, θz). K is necessarily a 6 × 6 stiffness matrix, which is often written as a set of four 3 × 3 submatrices:

=

K K KK K

tt tr

rt rr (3.8)

The stiffnesses are defined as follows:

θτ τ

θ= −

∂∂

= −∂∂

= −∂∂

= −∂∂q

; ; ;KF

qK

FK Kij

tt i

jijtr i

jijrt i

jijrr i

j

(3.9)

all evaluated at equilibrium q = qeqm. For a trapped sphere, all rotational terms will be zero, and the only nonzero terms will the diagonal terms of Ktt, that is:

=

0 0

0 0

0 0

K

K

K

K ttxxtt

yytt

zztt

(3.10)

with ≈ >K K Kxxtt

yytt

zztt (where z is the beam direction). However, for an extended par-

ticle, such as a prolate spheroid or an ellipsoid, the situation will be different. In the normal trapping configuration described earlier, an ellipsoid will orient with its longest axis parallel to the z axis, and its next longest axis parallel to the polarization direction (say, the x axis). In addition to the restoring forces, there will be restoring torques about each of the axes. Furthermore, there is a symmetry breaking due to the fact that the ellipsoid does not trap exactly at the focus of the trap, but is displaced due to the radiation pressure. This results in a rotational-translation coupling, that is, translations along x or y induce torques about y and x, respectively, while rotations about x or y induce forces parallel to y and x, respectively. The full stiffness matrix is given by [41]:

F=K(qqeqm)

K=KttKtrKrtKrr

Kijtt=−∂Fi∂qj; Kijtr=−∂Fi∂θj; Kijrt=−∂τi∂qj; Kijrr=−∂τi∂θj

Ktt=Kxxtt000Kyytt000Kzztt

Kxxtt∇Kyytt>Kzztt

772 Theory

=

=

=

=

0 0

0 0

0 0

;0 0

0 0

0 0 0

0 0

0 0

0 0 0

;

0 0

0 0

0 0

K

K

K

K

K

K

K

K

K

K

K K

K K

ttxxtt

yytt

zztt

trxytr

yxtr

rtxyrt

yxrt rr

xxrr

yyrr

zzrr

(3.11)

In the case of an oblate spheroid, with short axis perpendicular to both the beam axis (z) and polarization direction (x), symmetry dictates that any term relating rota-tions or torques about the y axis must be zero, that is, = = = 0K K Kyy

rrxytr

yxrt [41]. On

the other hand, for a prolate spheroid with long axis parallel to the beam axis, the only change to Eq. (3.11) required by symmetry is that = 0Kzz

rr [38].

2.3.2 Trap stability criteriaIn the low Reynolds number regime, the motion of a trapped particle is governed by the Langevin equation, which may be written:

ξ + = ( )tq Kq f L (3.12)

where ξ is the hydrodynamic resistance, or drag, matrix for the particle and fL(t) is the generalized stochastic force (i.e., it includes stochastic torques). fL(t) has zero mean, and variance proportional to ξ:

ξδ⊗ ′ = − ′( ) ( ) 2 ( )t t k T t tf fL LB

(3.13)

For a conservative system, the principle of equipartition can be employed, and the thermal energy equated with the elastic energy of the trap, to give:

= ⊗1

2

1

2k T I K q qB (3.14)

where 〈q ⊗ q〉 is the covariance matrix, and I is the unit matrix. Thus, in principle, the trap stiffness, K, can be determined from careful measurement of the covari-ances. However, in practice, K is rarely symmetric with the result that the system is nonconservative. For example, in Eq. (3.11), there is no physical reason for Kxy

tr to equal K yx

rt , or for K yxtr to equal Kxy

rt .When the system is nonconservative, equipartition fails and Eq. (3.14) does not

apply, making trap calibration difficult. A careful analysis of the thermal motion of vertically trapped nanowires illustrates the problem [54]. The asymmetric coupling between, say, translations parallel to the x axis and rotations about the y axis, leads to a cyclic motion, illustrated in Fig. 3.8. Since the force-field is nonconservative, the energy is path dependent and the bias toward cyclic motion shifts the energy away from its thermal equilibrium value, thus invalidating any attempt to relate it to the trap stiffness.

Ktt=Kxxtt000Kyytt000Kzztt; Ktr=0Kxytr0Kyxtr00000Krt=0Kxyrt-

0Kyxrt00000; Krr=Kxxrr000Kyyrr000Kzz-rr

Kyyrr=Kxytr=Kyxrt=0

Kzzrr=0

ξq˙+Kq=fL(t)

fL(t) ∇ fL(t′)=2kBTξδ (t−t′)

12kBT I=12 Kq⊗q

KxytrKyxrt

Kyxtr

Kxyrt

78 CHAPTER 3 Optomechanical microtools and shape-induced forces

Another way to examine trap stability is to look at the eigenvalues of the matrix K–1ξ. For exponential stability, the real part of each eigenvalue of K–1ξ should be positive [38,54]. In fact, if K is symmetric, the eigenvalues of K–1ξ will be real and correspond to the relaxation times for each of the vibrational modes of the trap. Equivalently, their reciprocals correspond to corner frequencies in the power spec-trum of each mode. However, when K is nonsymmetric, the eigenvalues may occur in complex conjugate pairs, in which case the time variation of the covariance be-comes oscillatory, leading to the cyclic motion described earlier.

Thus, if force probes are manufactured from shaped particles with uniaxial or ellipsoidal symmetry, it is clear from the earlier analysis that reliable force calibra-tion will be difficult or impossible, and an alternative approach is needed. For-tunately, the compound probes, or microtools, described in the following section satisfy this need.

FIGURE 3.8 Illustration of Nonconservative Motion in a Vertically Trapped Nanorod

Thermal fluctuations cause rotations and translations to occur. However, a translation perpendicular to the beam axis couples to a rotation about an axis perpendicular to the beam axis, and vice-versa. The arrows indicate the direction of the resultant probability currents.

792 Theory

2.3.3 Compound structures: microtoolsAs indicated at the beginning of this theory section, one aim of trapping low sym-metry particles is to achieve control over both their position and orientation, with a view to using the trapped particle to interact with some other, possibly biological system. An alternative approach to modifying the shape of individual particles is to form a composite structure, or microtool, of a form similar to the examples shown in Fig. 3.9.

The microtools shown in Fig. 3.9 share a common feature: each is constructed from a set of spherical handles, joined by thin struts. Each handle is held in an opti-cal trap—three handles are sufficient to allow full positional and orientational con-trol over the microtool. Each sphere has a diagonal 3 × 3 stiffness matrix, K i

tt, of the form given in Eq. (3.10). However, the stiffness matrix for the compound structure is more complex. Rigid body translations of the microtool arise from translations in each trap, as expected, but rigid body rotations, for example, about an axis parallel to the beam axis, also arise from individual translations. The construction of the stiffness matrix for the whole microtool is summarized by the following expres-sions [55]:

∑ ∑ ∑= = × = − × ×= = =

; ;1 1 1

K K K r K K r K rtitt

i

nc

ip

itt

i

nr

ip

itt

ip

i

n

(3.15)

Kitt

Kt=∑i=1nKitt; Kc=∑i=1nrip×Kitt; Kr=−∑i=1nrip×Kitt×rip

FIGURE 3.9 A Selection of Possible Microtool Geometries, Each Consisting of Trapped Spheres Joined by Narrow Cylinders

80 CHAPTER 3 Optomechanical microtools and shape-induced forces

with:

=

( )K K KK K

t c T

c r (3.16)

where the sums are taken over the n handles, and the rip are position vectors of the

handles in the microtool frame.The coupling matrix, Kc, is generally nonzero, but the structure of K in Eq. (3.16)

is such that this need not prevent K from being symmetric. However, if each handle-trap combination is equivalent, or if the individual trap stiffnesses differ only in mag-nitude, then translating the origin to the optical stress center will cause Kc to vanish. If, in addition, the individual traps are uniaxial (i.e., = ≠K K Kxx

ttyytt

zztt ), the handles are

large spheres and the microtool is planar and held horizontally, it is guaranteed that K can be diagonalized.

A consequence of this diagonalization is that, from the point of view of calibra-tion, the trapped microtool can be treated as if it were a single particle acted on by a single stiffness matrix. The position and orientation of the microtool can be deter-mined by tracking the positions of the spherical handles, and the covariances may readily be evaluated. The stiffness matrix elements then follow from application of Eq. (3.14), and nonconservative effects will not be a concern. The microtool effec-tively inherits the trapping properties of the sphere—a symmetric stiffness matrix and locally conservative force field.

Having outlined the basic theory underlying the trapping and force calibration of nonspherical particles and microtools, the following sections describe a range of experimental realizations of optically controlled microtools. Although there is some variety in the shapes and numbers of trapping handles employed in these microtools, the basic principles remain the same [55].

3 EXPERIMENTAL REALIZATIONS3.1 MICROTOOL FABRICATIONThe drive to miniaturize technology has spawned a host of methods for structuring matter on the nano- and microscale. Here we describe some of the techniques that have been used to fabricate optomechanical microtools. These techniques include directed assembly of components, 2D photolithography, direct laser writing, and un-conventionally, the selective culturing of silica shelled algae.

3.1.1 In situ directed assembly of componentsSome of the early work toward the fabrication of complex microtools explored the directed assembly of preformed components using optical micromanipula-tion. Individual components, such as microspheres and microrods [56,57], were functionalized in bulk with complementary biomolecules of a ligand-receptor system: biotin (microrods) and streptavidin (microspheres). The aim of this ap-proach was to exploit the high specific affinity exhibited by this ligand-receptor

K=Kt(Kc)TKcKr

rip

Kxxtt=Kyytt≠Kzztt

813 Experimental realizations

system to create a strong bond (relative to the picoNewton-scale forces exertable using optical tweezers) between functionalized components, once brought into contact [58].

Fig. 3.10 shows a TEM image of a high aspect ratio silica microrod produced by deposition into an alumina template containing pores of 300 nm in diameter and tens of microns (20–50 µm) in depth. After deposition, the template is dissolved and the microrods dispersed into water where they can be functionalized.

Fig. 3.11 shows an optical image of such a force transducer fabricated by James Grieve [59], possessing spherical handles which both can enable controllable optical micromanipulation and act as fiducial markers for robust nanometric video tracking of the structure’s position. The sharp tip is positioned remotely from the handles en-abling picoNewton scale forces to be measured at a location removed from the high intensities of the trapping beams.

This early approach proved challenging due to the nonspecific adhesion of the components to the sides of the sample cell, and also the difficulty in accurately posi-tioning the components to build a robust microtool. Nonetheless, such devices were built, their force response calibrated and were shown to measure forces in a series of proof of principle experiments [58,60]. Different assembly techniques are discussed in Chapter 2.

3.1.2 2D photolithographyPhotolithography is a microfabrication process enabling the microscale resolution patterning of a substrate. First the substrate is coated in a thin layer of photoresist.

FIGURE 3.10 A Silica Microrod of ∼5 µm in Length

Adapted from Grieve JA. Assembly & calibration of optically actuated tools. PhD thesis.

University of Bristol, 2011 [59].

82 CHAPTER 3 Optomechanical microtools and shape-induced forces

This is then illuminated through a preformed binary mask containing the desired pattern: therefore only parts of the photoresist are exposed where the light is trans-mitted, transferring the pattern to the photoresist. A series of etching steps then transfer the pattern to the substrate, and enable structures formed in the desired planar shapes to be released into solution. Fig. 3.12A–E shows a schematic and gives more detail of an example of this process. Fig. 3.12F shows examples of mi-crostructures fabricated using 2D lithography free-floating in solution. However, fabricating microtools using a single phase 2D lithographic process constrains them to a planar geometry, which as discussed earlier in the theory section, can limit their trapping stability without careful consideration of their design. Mul-tilayer lithographic techniques are also possible to build up 2.5–3D structures, however this is a significantly more complicated process requiring multiple pre-formed masks.

3.1.3 Direct laser writingDirect laser writing (DLW) is a high-resolution 3D photolithography technique which relies on the local solidification of a photoresist at the focus of a laser beam to “draw”

FIGURE 3.11 A Microtool Constructed by Directed Assembly

Adapted from Carberry DM, et al. Calibration of optically trapped nanotools.

Nanotechnology 2010;21:175501 [60].

833 Experimental realizations

structures with feature sizes down to ∼ 100 nm [61]. The technique is also referred to as two-photon polymerization because the polymerization of the photoresist is initi-ated by a two-photon process—that is, it requires the absorption of two photons at the same location simultaneously (or within a very short time of one another). This only

FIGURE 3.12 UV Lithography Process and Resultant Microtools

(A) The photoresist defines the 2D shapes to be fabricated. (B) The photoresist is developed and (C) reactive ion etching used to remove the SiO2 layer. (D) Sonication is used to release the resultant shapes. (E) SEM image of the 2D microtools still attached to the wafer. (F) Optical micrograph of 2D microtools free floating in water.

Adapted from Grieve JA. Assembly & calibration of optically actuated tools. PhD thesis.

University of Bristol, 2011 [59].

84 CHAPTER 3 Optomechanical microtools and shape-induced forces

occurs in a small volume at the center of the laser focus where the intensity is high enough. By sweeping the laser beam through the photoresist, optical quality 3D struc-tures can be reproducibly fabricated with voxel sizes down to ∼ 100 ×100 × 300 nm3. Chapter 2 gives more details about this technique.

Fig. 3.13A shows bespoke microtools fabricated using a commercially available DLW system (Nanoscribe Photonic Professional) [13,15]. Once an array of struc-tures has been fabricated on a surface, they are immersed in a droplet of water, gently disengaged from the substrate, and injected into a microscope sample cell, as shown in Fig. 3.13B [62].

FIGURE 3.13

(A) SEM images of microtools fabricated using direct laser writing, (B) optical images of the sample transfer process.

Part A: Adapted from Phillips DB, et al. An optically actuated surface scanning probe. Opt Exp 2012;20:

29679 [13] and Phillips DB, et al. Force sensing with a shaped dielectric micro-tool. Europhys Lett

2012;99:58004 [15]; Part B: Adapted from Phillips DB, et al. Fashioning microscopic tools.

SPIE Nanosci Eng; 2013:881029 [62].

853 Experimental realizations

3.1.4 Naturally occurring microtoolsDue to the complexity of the techniques to fabricate synthetic microtools described earlier, alternative ways of obtaining microstructures that can act as microtools have also been explored. One intriguing example is the use of living cells, such as silica shelled Diatom algae. Diatoms are unicellular photosynthetic algae which possess a silicified cell wall (also known as a frustule), and display a wide range of naturally occurring species-specific architectures [63]. Their potential as microtools to per-form force transduction and nanometric imaging has been explored [64–66], taking advantage of the relative ease of isolating and culturing species possessing these useful architectures.

Fig. 3.14 shows SEM images of the Diatom Nitzshia subacicularis. These are cigar-shaped algae approximately 30 µm in length, tapering to a tip diameter of ap-proximately 500 nm. They can be stably trapped and manipulated using two optical traps, and some conveniently exhibit two intracellular lipid globules of higher refrac-tive index which act as spherical “handles” for optical trapping, and also provide convenient tracking points.

3.2 3D TRACKINGThe ability to accurately track the position and orientation of microstructures with nanometric precision is crucial to transform them from mere floating objects into quantitative microtools. 2D tracking of spherical particles (i.e., motion in directions parallel to the focal plane of a microscope) can be achieved by performing feature tracking on high-speed camera images. Algorithms designed to locate the center of mass or the center of symmetry of symmetrical objects can return 2D coordinates with significantly subpixel precision (approximately corresponding to nanometers) [67]. The fact that this precision is inherently subwavelength and therefore well be-low the diffraction limit sometimes causes incredulity. However, the diffraction limit is not relevant here as the aim of 2D video tracking in this context is not to differ-entiate between two closely spaced points (the minimum separable distance which would be diffraction limited), but instead to determine the centroid of a large object (covering several pixels). This problem is analogous to determining the peak of a 2D

FIGURE 3.14 Optical (left) and SEM (right) Images of Diatom Algae

Adapted from Phillips DB et al. Surface imaging using holographic optical tweezers. Nanotechnology

2011;22: 285503 [66] and Phillips DB et al. Position clamping of optically trapped microscopic

non-spherical probes. Opt Exp 2011;19:20622–20627 [65].

86 CHAPTER 3 Optomechanical microtools and shape-induced forces

histogram, which evidently can be achieved to a precision of subhistogram-bin-width by fitting a smooth function to the data, and a similar approach is taken here.

The precise tracking of nonspherical objects from camera images is much more challenging than the tracking of their spherical counterparts. For example, rotational motion out of the focal plane (i.e., about an axis parallel to the fo-cal plane) causes the image of the object to change in a nonsymmetrical man-ner, which artificially couple measurements of lateral position to orientation. Other couplings also appear as the object moves and rotates in 3D, and these significantly deteriorate even 2D tracking precision. Therefore, it is prudent to construct microtools with spherical tracking points (which conveniently can also double as trapping handles), facilitating accurate tracking of these more complex structures.

If possible, 3D tracking is preferential, as it is only then that the true 3D position of the probe tip can be inferred from the 3D position and orientation of the rigid structure. By using three noncolinear tracking points, all six degrees-of-freedom of the rigid structure can be uniquely determined [15]. 3D tracking of spherical ob-jects can be achieved in several different ways: by correlating prerecorded image stacks captured at different heights [68], by observing the position of a laser beam focused through the sphere with a quadrant photodiode [8], or using stereomicros-copy [69–71].

Stereomicroscopy is most compatible with holographic optical tweezers as it does not use the tracking beams to extract position information, and does not require any calibration images to be recorded. Fig. 3.15 shows a schematic of a dual illumination stereomicroscope system integrated with a holographic optical tweezers system, developed by Bowman and coworkers [70,71]. By simultane-ously illuminating the sample from two different directions 60 degrees apart, two views of the sample overlap on the camera (one from each direction). As spherical objects look approximately the same regardless of viewing direction, 2D tracking of the apparent center of mass (or symmetry) in each of these images can be combined using parallax to recover the three-dimensional coordinate of a spherical object. However, the overlapping images must be separated in order to achieve this. Separation can be accomplished by either using color channel (il-luminating from different directions with different colors) [69], or using simple additional optics: the two images form separate points in the Fourier plane of the sample, therefore a pair of oppositely tilted prisms in the Fourier plane can be used to separate these images so they appear adjacently on the camera, as shown in Fig. 3.15. It is also possible to use an SLM to separate the images [72,73], which offers the possibility to dynamically change the depth-of-field and in-crease it from a few microns to 10–100s of microns [74].

3.3 3D OPTICAL CONTROLA key requirement for the accurate control of microtools is the ability to generate multiple optical tweezers that can be dynamically reconfigured in three dimensions

873 Experimental realizations

“on the fly.” Crucially, this allows control of their motion to be handed over to a com-puter by implementing feedback algorithms: their movement can be choreographed with nanoscale finesse.

There are a variety of possible techniques to generate reconfigurable optical traps, for example, holographic optical tweezers [75], time-shared traps [76], and the generalized phase contrast method [77]. Holographic optical tweezers use a phase-only spatial light modulator which acts as a reconfigurable diffractive opti-cal element that can split an incident laser beam into multiple beams that, when focused into the sample, form separate optical traps [78]. The phase pattern dis-played on the SLM controls the number, position, shape, and relative power of the resulting three-dimensional array of traps. Typically, such phase patterns can be reconfigured at video rates of up to ∼200 Hz (although kiloHertz rate switching

FIGURE 3.15

(A) Schematic of a holographic optical tweezers system with a stereoscopic tracking arm. (B) Left eye and right eye views of an optically trapped microtool.

88 CHAPTER 3 Optomechanical microtools and shape-induced forces

has also been demonstrated [79]), giving dynamic control of microtool position and orientation.

There are a variety of algorithms that can be used to calculate the phase pattern required to generate a particular optical trap configuration. A simple and powerful algorithm is known as “gratings and lenses” or “direct superposition” [80]. The SLM is typically positioned in the Fourier plane of the sample. The gratings and lenses algorithm takes advantage of the conjugate relationship between position in the sample plane and direction in the Fourier plane. Therefore, the lateral transla-tion of an optical trap within the sample plane corresponds to a change in direction of the corresponding light beam in the Fourier plane at the SLM. The direction of a light beam reflected from an SLM can be controlled by applying a phase tilt across its surface. Therefore each trap position corresponds to a particular phase tilt across the SLM. Calculating the complex sum of these phase tilts yields a phase pattern that produces traps at the desired locations in the sample. Using this method additional unwanted traps in the sample may also be produced (known as ghost traps), however, these may be suppressed if necessary. This concept can also be extended to incorporate quadratic lens phase profiles that translate the axial position of the traps if necessary (in the direction normal to the focal plane). By implementing the gratings and lenses algorithm on graphics processors, phase patterns can be calculated considerably faster than the update rate of SLMs, giving virtual real-time 3D control (at ∼200 Hz) of the trapping beams [81]. Fig. 3.15 shows a schematic of a typical holographic optical tweezers set-up built around an inverted microscope.

4 APPLICATIONSMuch of the work toward applications of this technology has focused on the develop-ment of novel imaging techniques, and in particular a new form of scanning probe mi-croscope (SPM). The concept is to scan a microtool over the surface of a sample while precisely tracking the position of its sharp tip, thus revealing a nanometrically resolved image of the surface. Using an optically trapped microtool rather than the conventional SPM approach of using a piezocontrolled atomic force microscope (AFM) tip offers several new opportunities: softer samples can be imaged as the optical probe exerts much lower forces on the surface of the sample—down to single picoNewtons or 100s of femto-Newtons which is two to three orders of magnitude lower than AFM. Also sample access is improved as the sample can be approached from arbitrary directions, and the device can be operated inside a sealed microfluidic chamber. Finally, by using holographic optical tweezers it is relatively straight-forward to potentially increase the number of independently operating scanning probe tips. In parallel we also note that there have been a number of experiments using optically trapped microspheres to im-age surfaces—a powerful complementary technique [4].

There have been several proof-of-principle surface-imaging experiments using different types of microtools. Fig. 3.16 shows one of the first examples, recorded

894 Applications

FIGURE 3.16 Surface Images of the Soft Algae “Pediastrum” Recorded Using an Optically Trapped Diatom

(A) The 3D surface image of the spike encircled in (B) and shown in close-up in (C). Optical micrograph (B) also shows an optically trapped Diatom. (D) A 3D surface image following a scan of the side wall of a Pseudopediastrum unit cell. (E) A bright field confocal image and (F) optical micrograph showing similar regions to (D).

Adapted from Phillips DB, et al. Surface imaging using holographic optical tweezers. Nanotechnology

2011;22: 285503 [66] and Phillips DB. Applications of closed-loop feedback control with holographic

optical tweezers. PhD thesis. University of Bristol, 2012 [82].

90 CHAPTER 3 Optomechanical microtools and shape-induced forces

using the Diatom algae described earlier as an imaging probe. In this work, the relative depth of each pixel in the image was inferred by stepping an optically trapped Diatom toward the sample until the body of the Diatom is observed to translate or twist due to its tip’s interaction with the sample. Tracking could be achieved using image comparison rather than full 3D tracking, although the requirement to step back and forth to measure each pixel renders a low imaging rate. Nevertheless, this first example demonstrates the potential of such a technique to reconstruct 3D images of objects inside a sample to a resolution of 320 nm laterally (limited by the tip radius), with an 80-nm depth resolution [66,82].

A second set of experiments considered the use of synthetic microtools fabricated using direct laser writing, shown in Figs. 3.1, 3.17, 3.18. The 3D translation and rota-tion of these objects was tracked in 3D as described earlier using stereomicroscopy, and so the trapping characteristics of the microtool could be quantitatively calibrated. In this work, an understanding of shape-induced forces was leveraged to improve the performance of the microtool. Namely, the trapping points were cylindrical, and so the tool exhibited an anisotropic trapping stiffness profile that was very soft in the direction normal to the sample, resulting in extremely low normal forces exerted on the sample (Fig. 3.17). Using this approach the tip could be scanned directly across the surface, significantly increasing the imaging rate. In addition, by monitoring the position of the microtool in real-time, feedback was used to modulate the trapping laser positions, thus maintaining a constant average force of only 140 fN on the sur-face as it was scanned. Fig. 3.18 shows a line-scan of a calibration surface recorded using this technique. Here a lateral resolution of 200 nm and a depth resolution of 11 nm was demonstrated.

FIGURE 3.17 Schematic Showing How the Volume of Space Explored by the Probe’s Tip Varies With Trapping Position and Active Trapping Feedback

(A) When the traps overlap the end caps of the cylindrical handles, the trapping stiffness is maximized, yielding the smallest tip thermal volume. (B) When the traps are positioned 3 µm inside each end of the cylinders, the thermal volume expands. (C) Reducing the trapping power by ∼50% further increases the thermal volume. In each case, the tip thermal volumes shown are expanded by a factor of 60 relative to the probe sketch.

Adapted from Phillips DB, et al. An optically actuated surface scanning probe.

Opt Exp 2012;20: 29679 [13].

914 Applications

A third experiment took shape optimization a step further and incorporated feed-back control directly into the structure itself. By creating conically shaped trapping points with a tapering radius, a flat force response can be engineered that exhib-its a constant optical restoring force regardless of axial displacement, as shown in Fig. 3.19. This means that as the device is scanned across the undulations on a sur-face, it always exerts the same average normal force on the sample with no need to provide feedback on the trap positions. A caveat is that when riding over a bump in the surface, the system must relax to its new configuration, and this relaxation time depends upon its friction tensor. Fig. 3.20 shows a surface image recorded using this microtool.

FIGURE 3.18 Recording Surface Topography Using an Optically Trapped Microtool

(A) and (B) show the trajectory of the probe tip as it approaches the sample, and scans laterally over steps of different depth to illustrate the height resolution. (C) A scan over corrugated part of another test sample. (D), (E), and (G) show SEM images of the test sample. (F) Shows a stereo-microscope pair of images of the probe and sample prior to the start of the experiment.

Adapted from Phillips DB, et al. An optically actuated surface scanning probe.

Opt Exp 2012;20: 29679 [13].

92 CHAPTER 3 Optomechanical microtools and shape-induced forces

FIGURE 3.20 A Constant force (Exerted on Sample) Surface Image Recorded with a Microtool With a Flat Optical Restoring Force

(A), (B), and (C), SEM images of tapered constant force probes. Vertical fabrication is used to optimize the tip sharpness. (D) Optical image of the constant force probe scanning across a calibration sample. (E) Surface topography of the side of the calibration sample recorded during the scan. (F) SEM image of the calibration sample. (G) Data from (E), viewed in 3D for comparison with (F).

Adapted from Phillips DB, et al. Shape-induced force fields in optical trapping.

Nat Photonics 2014;8: 400–405 [16].

FIGURE 3.19 A Microtool Engineered to Exhibit a Flat Optical Restoring Force

(A) Calculated optical force exerted on a tapered cylinder. (B) Experimental force–displacement profile of a particle with a linear taper, shown schematically in (C) and in the optical micrograph in (D), which was fabricated using direct laser writing. In (D) the red circles indicate trap positions.

Adapted from Phillips DB, et al. Shape-induced force fields in optical trapping.

Nat Photonics 2014;8: 400–405 [16].

935 Conclusions and future prospects

5 CONCLUSIONS AND FUTURE PROSPECTSIn this chapter, some of the recent progress in the emerging field of optomechani-cal microtools has been reviewed. The underlying theory describing how to predict the trapping characteristics of arbitrarily shaped microstructures has been outlined. Rigorous methods of microtool calibration and characterization have also been de-scribed. The experimental techniques that have been used to fabricate, optically con-trol, and track such devices have been introduced. Finally, some of the proof-of-principle experiments pointing toward new applications, such as force and torque measurements and surface imaging have been described.

Although imaging and force measurement has been the focus of this review, the flexibility of fabricating arbitrarily shaped structures, combined with an understand-ing of how they will behave in optical and hydrodynamic fields promise a host of new advances in the near future. For example, Fig. 3.21 shows a prototype optical rotator, designed to grasp and rotate objects (such as cells) about an axis parallel with the focal plane of the microscope—a reorientation that is challenging to achieve with current technology. This has the potential to lead to a new form of cell tomog-raphy, allowing reconstructions of the internal structures of cells with isotropic high resolution. Adhesion of streptavidin coated microtools to biotinylated cells has also recently been demonstrated [83]. In addition, the small cross-sectional dimensions of some microtools lead to the possibility of making flexible tools, which might be used to corral particles or cells, leading to novel forms of interaction. Fig. 3.22 shows example probes made from the polymer SU8 using electron beam lithography; the flexible links are 150-nm wide and can be deformed by manipulating the end sup-ports in optical tweezers [84].

FIGURE 3.21 A Prototype Optically Trapped Rotator, Capable of Rotating Objects About an Axis Parallel to the Microscope Focal Plane

(A) SEM of the optical rotator. (B) Optical micrographs showing the rotator in operation.Adapted from Phillips DB, et al. Fashioning microscopic tools. Proc SPIE 2013;8810:881029 [62].

94 CHAPTER 3 Optomechanical microtools and shape-induced forces

Beyond the examples discussed in this chapter, in parallel there have been a host of impressive developments by other groups, several of which are also detailed in the other chapters of this book. Although this field is still in its infancy, the rapid devel-opment of fabrication techniques and experimental protocols promises an exciting future.

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99

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00004-5Copyright © 2017 Elsevier Ltd. All rights reserved.

Anatolii V. Kashchuk, Ann A.M. Bui, Shu Zhang, Antoine Houillot, David Carberry, Alexander B. Stilgoe, Timo A. Nieminen, Halina Rubinsztein-Dunlop

The University of Queensland, St Lucia, Brisbane, QLD, Australia

CHAPTER OUTLINE

1 Introduction ........................................................................................................ 992 Optical Angular Momentum ................................................................................ 1003 Principles of Design .......................................................................................... 102

3.1 The Importance of Symmetry ..........................................................1023.2 Discrete Rotational Symmetry With p = 2 .........................................1043.3 Discrete Rotational Symmetry With p > 2 ........................................1063.4 No Rotational Symmetry (p = 1) ......................................................1093.5 Using Reflection to Generate Torque ................................................1093.6 Controlling Reflection .....................................................................110

4 Computational Modeling .................................................................................... 1165 Fabrication ....................................................................................................... 1166 Applications ..................................................................................................... 117

6.1 A Case-Study: Pinned Cross-Rotors ..................................................1187 Conclusions ...................................................................................................... 123References .............................................................................................................. 123

1 INTRODUCTIONThe question “what is an optically driven micromachine?” is a convenient place to begin. A broad definition of “machine” could include even ordinary microspheres of silica, polystyrene, etc., which are commonly trapped in optical tweezers and used as “handles” for exerting or measuring of forces. A narrower definition of “machine” might be much more restrictive. Here, we will opt for a more restrictive definition, and only consider the narrow class of optically driven rotating micromachines. These are characterized by the capability of motion—rotational motion—while the ma-chine as a whole is held in place, most commonly by an optical trap, or possibly incorporated in some larger device, such as a microfluidic device. Various fabricated microtools [1–5] that can be usefully manipulated in optical traps fall in the wide range between the rotating machines we will consider here and simple microspheres,

Optically driven rotating micromachines 4

100 CHAPTER 4 Optically driven rotating micromachines

but we will not consider them here. With our limited scope here, we will not be able to provide a comprehensive review; even a decade ago, a brief review of the topic [6] could amount to almost 100 references.

We will assume that our readers are familiar with optical tweezers and the prin-ciples of optical forces, but will provide an introduction to optical angular momen-tum and torques. Readers seeking further details about computational approaches to modeling or simulating optical tweezers can refer to [7–11]. We will then proceed to outline general principles of design, construction, and operation of micromachines. Finally, we consider applications, and present a prototypical demonstration of a ro-tating machine incorporated in a microfluidic device.

2 OPTICAL ANGULAR MOMENTUMThe fact that light can carry angular momentum follows naturally from light carrying linear momentum. If a beam of light has momentum density p(r), it will possess a corresponding angular momentum density

= × ( )L r p r .

Both the momentum and angular momentum densities can be integrated over the beam to find the total quantity. If the beam is assumed to be infinitely long, such integrals are typically infinite, and it is then more convenient to integrate instead momentum and angular momentum flux densities over, for example, a cross-section through the beam, or a closed surface surrounding a region of interest.

If we consider a ray of light in a homogeneous isotropic medium, it is analogous to the trajectory of a force-free object in classical mechanics. This leads to two inter-esting observations. First, the vector component of the angular momentum of the ray parallel to the momentum must be zero. Second, if we choose an origin to take mo-ments about which is on the ray, the angular momentum is zero. These are in conflict with common knowledge in quantum mechanics that light carries ±ħ spin angular momentum per photon, about the direction of propagation. Thus, the spin angular momentum doesn’t arise from the moment of the linear momentum, and is inde-pendent of our choice of origin about which to take moments. It is therefore termed intrinsic angular momentum. Thus, we must include our spin as additional contribu-tion to the angular momentum, and our total angular momentum J can be written as the sum of spin and orbital components, S and L, respectively, as

= +J L S

with similar expressions whether we are working with densities, flux densities, or integrated totals of either densities or flux densities.

While we noted that the orbital angular momentum of a ray about its direction of propagation must be zero, this is not the case for a beam. A simple picture of a focused beam is as a bundle of converging rays. If the rays do not pass through the

L=r×p(r).

J=L+S

1012 Optical angular momentum

beam axis, but are skew to it, they will have a component of momentum normal to the beam axis that will give a nonzero contribution to the angular momentum about the beam axis. If we consider a converging cone of rays, in which the rays all pass through a ring, with the same handedness, we have a simple ray model of a Laguerre-Gauss beam [12–14], which is commonly used to rotate optically driven micromachines due to this orbital angular momentum about the beam axis [13,14]. Interestingly, for a beam, such as this, the total linear momentum normal to the beam axis is zero, and the integral of the orbital angular momentum flux parallel to the beam axis becomes independent of a shift of the origin about the beam axis, despite the density and flux density being dependent on it. This has been termed “intrinsic orbital angular momentum” [15] to distinguish it from the usual case where the inte-gral is dependent on the choice of origin.

We do not need to consider explicit formulae for the calculation of spin and or-bital angular momenta (but these can be obtained in many works, e.g., [16–19]; note that the integrals of the flux densities are easiest to calculate in the far field, since the flux densities are tensor quantities). What we need to consider is the impact of the difference between spin and orbital angular momenta on the design and operation of optically driven micromachines. First, the spin that can be carried by a beam is lim-ited by the maximum of ħ per photon. Extending this to a classical picture of light, this implies a maximum spin flux of

ω=S P /

for a beam of power P and optical frequency w. If the beam is focused, this is further reduced since—adopting the bundle of converging rays as earlier—only the com-ponent of the spin parallel to the beam axis can contribute to the total flux [20]; this is analogous to the reduction in linear momentum flux due to the convergence of a beam [21], which was exploited to generate axial gradient forces in optical tweezers. There is no such limit to the orbital angular momentum, since the moment arm can be, in principle, made arbitrarily large. However, an arbitrarily large moment arm requires an arbitrarily large beam, for angular momentum about the beam axis. Thus, the size of the focal spot of a beam limits the orbital angular momentum about the beam axis. Since the spin is dependent on the frequency, and thus the wavelength, how tightly the beam can be focused is limited by its angular momentum content. Together, these limit how much angular momentum can be delivered to a given focal volume by a beam of a given power [22]. This limits the delivery of angular momen-tum to small micromachines. Despite this, small micromachines will often rotate at higher rates than larger micromachines driven by larger beams with more angular momentum, since viscous drag will scale with the size of the machine cubed (at low Reynolds numbers).

The consequence of the difference between spin and orbital angular momenta is that the spin angular momentum depends on the polarization of the beam, and useful transfer of this angular momentum to a micromachine requires anisotropy—the machine can be constructed of a birefringent material [23–25] or we can use

S=P/w

102 CHAPTER 4 Optically driven rotating micromachines

shape birefringence [26]. Changing the direction of propagation of the light, by refraction or reflection, on the other hand, can usefully transfer orbital angular momentum.

There are continuing difficulties and controversies in the field of electromagnetic angular momentum. There are differing claims of the correct expressions for the spin and orbital components of the angular momentum. What is known is that the moment of the Poynting vector, × ×( ) / cr E H , where E and H are the electric and magnetic fields respectively, and c is the speed of light in free space, can be integrated over all space to give the total angular momentum—except for special cases, such as an infinite circularly polarized plane wave, where it yields zero. On the other hand, one can obtain the canonical stress tensor and a matching angular momentum tensor via Noether’s theorem [18–19], which can be divided into spin and orbital components [18–19], disagreeing with the earlier result of zero angular momentum for the infinite plane wave. The conditions under which the two approaches yield the same result are the same as the conditions under which the canonical stress can be transformed into the symmetric stress tensor [18]. For electromagnetic fields, such as beams of finite width, both approaches work—one is free to choose to either integrate the moment of the Poynting vector, or to calculate orbital angular momentum using the orbital angular momentum operator, and the spin from the polarization; the most convenient should be used. For special cases, such as plane waves, it is easiest to calculate the spin from the polarization, since the orbital angular momentum about the direction of propagation is zero.

The other major dispute concerns the gauge freedom and Lorentz invariance of the division into spin and orbital components. For fields, such as monochromatic beams, this is a nonissue, since a gauge-independent separation is straightforward [16], and the choice of a monochromatic beam implies the choice of a specific iner-tial reference frame (the beam would not be monochromatic in a relatively moving frame). Therefore, this is not a problem in practice.

3 PRINCIPLES OF DESIGN3.1 THE IMPORTANCE OF SYMMETRYA key feature of the design of optically driven micromachines is rotational sym-metry—a rotationally symmetric object will not change the angular momentum per photon about its symmetry axis. A birefringent micromachine breaks any such ro-tational symmetry at the molecular level (unless the optic axis is aligned along the symmetry axis). A machine driven by orbital angular momentum must break this symmetry by its shape, usually through the adoption of a shape with discrete, rather than continuous, rotational symmetry. As shown in Fig. 4.1, forces due to reflection and refraction exerted on a rotationally symmetric object pass directed through the symmetry axis, and produce no torques about that axis. This is simply explained as a consequence of forces due to reflection and refraction at a surface involving no change of momentum along the surface.

r×(E×H)/c

1033 Principles of design

To examine the impact of discrete rotation symmetry using some useful math-ematic tools, we can introduce the representation of a monochromatic electromag-netic field as a superposition of modes with well-defined angular momentum and orthogonality of angular momentum and power. That is, we seek a representation of fields such that

∑ ψ= a( ) ( ) ,i ii

E r r

where ai is the amplitude of the ith mode, and ψ(r)i is a function specifying the field of the ith mode. The two most useful choices are the Laguerre-Gauss (LG) modes, which are solutions of the scalar paraxial wave equation (and therefore only give the orbital angular momentum directly, requiring an additional description of polarization to also include spin) and the vector spherical wavefunctions (VSWFs), also known as the TE and TM multipole fields, which are solutions of the vector Helmholtz equation and therefore give the combination of spin and orbital angular momenta [16,27,28]. The key to the usefulness of modal descriptions like these in a discussion of angular momentum is that the azimuthal variation of the field is given by

φimexp( )

where m is the mode index describing variation about the z-axis. Where the z-axis coincides with the beam axis, this results in LG modes carrying mħ orbital angular momentum per photon about the beam axis, and VSWFs mħ total angular momen-tum. The relationship between LG beams and VSWFs has been explored in [29–31]. Strictly speaking, the VSWFs should be used as a basis set of modes when consider-ing tightly focused beams, but the LG modes provide a convenient basis for an ap-proximate heuristic approach, as long as the transfer of spin can be neglected.

E(r)=∑iai(r)i,

exp(imφ)

FIGURE 4.1 Reflection and Refraction Forces Acting on Plane and Curved Surfaces

In both cases, the forces are normal to the surface, with the refraction force directed toward the lower refractive index. Where the curved surface forms a rotationally symmetric shape (i.e., circular in cross-section), both the reflection and refraction forces are directed through the center, and moments of these forces about that center are zero.

104 CHAPTER 4 Optically driven rotating micromachines

The usefulness of such descriptions of beams results from the fact that an object with pth order discrete rotation symmetry about the z-axis couples an incident mode with m = m0 to modes with

= +m m pk,0

where k is an integer. Thus, an object with no rotational symmetry (i.e., p = 1) can couple all modes, while continuous rotation symmetry (p = ∞) doesn’t alter the angular momentum of modes.

The strength of the coupling depends on the details of the structure. It is also limited by the angular momentum content of the modes. A mode of angular mo-mentum m has a minimum radius of approximately, λ π=m k m/ / (2 ), where λ is the wavelength of the light in the surrounding medium [9]. This is, of course, related to the limit on how much angular momentum can be delivered to a given area [22], discussed earlier. Unless the modes overlap sufficiently that the structure can couple light between them, coupling will not be possible.

3.2 DISCRETE ROTATIONAL SYMMETRY WITH p = 2Since the VSWF description includes polarization, we can consider the effect of symmetry on polarization. We can consider arbitrary elliptical polarization (includ-ing circular and linear) as superpositions of both possible circular polarizations. Since these differ in spin angular momentum by 2ħ, a beam produced by focusing a beam with orbital angular momentum given by m0 (e.g., an LG beam with m0) will give a VSWF description with

= ±m m 1.0

For the important case of a Gaussian beam (m0 = 0), this gives = ±1m . Since the relevant modes are separated by 2, the symmetry of importance is p = 2.

With this symmetry, it is possible to use spin angular momentum as the source of torque. This has two major advantages. First, it is possible to operate such a micro-machine in three distinct modes: plane polarized light results in an angle-dependent torque acting to align the machine with the plane of polarization. Control of the plane of polarization allows the orientation to be controlled, and the machine can be turned by a desired angle. If the plane of polarization can be continuously ro-tated, the machine can be driven in a constant-speed, variable-torque mode. If the light is circularly polarized, the torque is independent of the angle, and continuous rotation—a variable-speed constant-torque mode—results [24,32].

Second, it is possible to determine the torque and the angular position of the ma-chine by measuring the polarization state of the transmitted light [32,33]. This can be useful for feedback and control, and allows much simpler use as a quantitative probe for torques.

It is possible to use orbital angular momentum, instead of or in addition to spin angular momentum. The first of the earlier advantages—simple multimode

m=m0+pk,

m/k=mλ/(2π)

m=m0±1.

m=±1

1053 Principles of design

operation—can still be achieved, but the second one of easy quantitative measure-ment of torque is lost, as the optical measurement of orbital angular momentum is much more difficult. Notably, methods that work well on the macroscale optical table [34] are very difficult to transfer successfully to the microworld of optical tweezers [35]. Sometimes, where both spin and orbital angular momenta contribute, the total torque can be estimated from the spin torque and the rotation rate [36,37].

This symmetry characterizes uniaxial birefringent materials on a microscopic scale (and can provide a convenient approximation for many biaxial materials). It was recognized early in the field of optical rotation that birefringent materials were very promising for micromachines [23,24,32]. If the birefringence is high, high torque efficiencies, with angular momentum transfers greater than 1 (in units of ħ per photon), are possible with modest thicknesses (less than 2 µm for calcite) [24]. Weakly-birefringent materials will require a greater thickness to effect the same change in the polarization of the driving light. The difficulties lie in production and fabrication, and chemical interaction with the environment.

Natural crystalline materials can be used. Vaterite (a form of calcium carbonate) nanocrystals will self-assemble into spherulites [38–40] on the order of a micron or a few microns in size. Vaterite microspheres have been very widely used in optical ro-tation due to their ease of production—simple shake-in-a-test-tube chemistry. Their birefringence is high [39], and consequently high torques are easily obtained. In ad-dition, their spherical shape is convenient for many applications: (1) the viscous drag torque D experienced by a sphere of radius a rotating with angular speed Ω in Stokes flow has a simple analytical form, πη= ΩD a8 3 , where η is the dynamic viscosity, (2) the surrounding fluid flow field is also simple, and (3) their spherical shape means that shape effects on orientation do not interfere with orientation—their symmetry in the optical sense is determined solely by their birefringence. Their chief disad-vantages are: (1) they are polydisperse, with the details of manufacture determining their size range, and consequently their exact size is unknown without measurement, which is difficult to perform with very high precision using an optical microscope, (2) they are not exactly spherical, which affects the accuracy of the Stokes drag formula and complicates size measurement, and (3) they dissolve rapidly in acidic environments, and many techniques commonly used for attachment of biomolecules do not work. Overall, judging by their widespread use, the advantages of vaterites outweigh their disadvantages. However, vaterites can be coated with silica [40] (and possibly other coatings), preventing destruction by an acidic environment, and allow-ing common functionalization protocols to work very well.

Other birefringent materials have been used. Various shapes have been fabricated from quartz [41] and birefringent polymers [23]. Small-scale structuring can be used to make a material birefringent; this can be done as part of the fabrication process of the micromachine [26,42]. One interesting possibility is the use of liquid crystals, which can form highly spherical droplets [43–46].

However, it is not necessary to use birefringent materials to satisfy the goal of p = 2 rotational symmetry; this can also be achieved by the overall shape of the ob-ject. The orientation of such objects by linearly polarized light was observed early

D=8πηa3Ω

106 CHAPTER 4 Optically driven rotating micromachines

[47,48], and continuous rotation in circularly polarized light followed shortly [33]. Two very simple shapes provide this symmetry: elongated objects, and flattened ob-jects. While they can still possess rotational symmetry about their long and short axes respectively, they have p = 2 symmetry about their short (for elongated objects) and long axes (for flattened objects). Elongated objects have a distinct disadvantage: they tend to align along the beam axis [49], thus presenting a rotationally symmetric cross-section to the beam (unless they are very small; elongated nanoparticles can still align with the polarization direction [50–52]). It is, in principle, possible to use two traps, one to trap each end of the elongated object, but a strong tendency to escape one of the traps and align along the axis of the other is usual. For practical applications, flattened objects are easier. Possible objects include polystyrene micro-spheres that have been flattened, wax discs [53], or microfabricated discs. One pos-sibility is to use a larger particle (ideally spherical, or possibly elongated for orienta-tion along the beam axis) with an elongated inclusion. If the elongated inclusion is low-index compared to the rest of the particle (possibly a cavity or hole), it will act as a negative uniaxial birefringent particle, and align normal to both the beam axis and the plane of polarization. This has the inconvenience of being laterally repelled by the beam, and will be trapped off-center; this can be prevented by using, for example, two hollow channels through the larger particle [42].

Objects, such as these affect the polarization of the light—they possess shape bi-refringence—and positions and torques can be measured via the polarization. How-ever, torque efficiencies are typically low, often well below 0.1ħ per photon [33]. Once the small dimension ceases to be small compared to the wavelength, the effect on the polarization is reduced. Smaller objects have a higher effective birefringence per unit volume, but smaller volumes, and do not deliver high efficiencies. This, combined with the more complex drag and fluid flow resulting from nonspherical shapes, are probably responsible in large part for the limited practical use of simple shapes, such as these.

Higher torque efficiencies can be obtained by using structured illumination, such as the twin traps described earlier for the (attempted) rotation of an elongated par-ticle, or elliptical focal spots matching the cross-section of the particle.

One interesting solution to the problem of elongated objects aligning with the beam axis is to use it to drive the rotation. If a number of beams are delivered from different directions in a horizontal plane, intersecting at a common point where the particle will be trapped, the particle can be controlled to lie along different beams by switching them on and off individually [54].

3.3 DISCRETE ROTATIONAL SYMMETRY WITH p > 2Optically driven micromachines with higher order discrete rotational symmetry, with p = 3 and greater, have been demonstrated. Since these do not couple the modes responsible for the two handednesses of circular polarization (which are separated by a difference in m of 2), such micromachines primarily operate based on orbital angular momentum. Standard techniques used to generate beams carrying orbital angular momentum about their axis, such as LG beams, provide us with a very useful

1073 Principles of design

heuristic for developing designs of such micromachines. On-axis optical phase ele-ments (also called diffractive optical elements, or holograms) can imprint the desired azimuthal phase variation on a Gaussian beam, or transform the angular momentum content of an LG beam. Thus, torques are exerted on such optical elements, and suit-able microscopy versions can be used as optically driven micromachines.

Some examples are shown in Fig. 4.2. All three have discrete rotational symmetry of p = 8. Since these are designed as phase objects, the difference in thickness between the thickest and thinnest parts of the structure should be, such as to produce a full-wave phase shift. The thickness of the thinnest parts can be zero, in which case the structure is shaped like a cross or star. The first two examples are mirror symmetric, and thus do not experience torques in linearly polarized Gaussian beams—they scatter symmetri-cally from the incident = ±m 10 modes to modes with = ± ±m 7, 9 , etc. While this might appear to be a disadvantage (and is, for some applications), it allows equal torques in both directions by driving the machine with LG beams of opposite handedness. For these two machines, the optimal conditions should be driving them with beams having m0 = 4 or m0 = −4, which will couple strongly to modes with m = −4 and 4 respective-ly. This coupling should be relatively efficient, since the modes with = ±m 4 can be the same size, maximizing the mode overlap and hence the coupling. Computationally

m0=±1m=±7,±9

m=±4

FIGURE 4.2 Optically Driven Micromachines based on Optical Phase Elements for Transforming the Orbital Angular Momentum of Laser Beams.

All three have p = 8. The structures in figures (A) and (B) are also mirror symmetric, and will transform an incident Gaussian beam equally to left- and right-handed modes, generating zero torque. The portion of the Gaussian beam passing through the center section will not generate any torque, so ideally the beam should be large enough to just illuminate the entire rotor. These can be rotated using incident Laguerre-Gauss beams, and can be rotated with equal ease in each direction by changing the handedness of the incident driving beam. The structure in Figure (C), on the other hand, is chiral rather than mirror symmetric, and can be driven by a Gaussian beam.

108 CHAPTER 4 Optically driven rotating micromachines

modeling indicates that torque efficiencies of about 0.4 can be achieved with such p = 8 rotors, while about 0.3 can be achieved in practice with short-of-perfect fabrica-tion [55]. Efficiencies of 0.2 have been achieved for p = 4 rotors, driven by beams with

= ±m 20 [37]. The torque efficiency is related to the torque τ by

τ ω= Q P / ,T

where QT is the torque efficiency, P is the beam power, and w is the optical frequen-cy. The torque efficiency can be interpreted as the transfer of angular momentum per photon, in units of ħ. Similarly, the force F can be given in terms of a force efficiency QF, such that

=F nQ P C/ ,F

where n is the refractive index of the surrounding medium, and c is the speed of light in vacuum. The force efficiency is the transfer of linear momentum per photon, in units of ħk.

The rightmost device in Fig. 4.2 is chiral, rather than mirror-symmetric, and can be usefully rotated when driven by a Gaussian beam. In addition, if driven by inci-dent LG beams, it will generate higher torque efficiencies for one incident handed-ness. The penalty paid for this is that it will not rotate with equal torque in both directions, which is a reduction in flexibility in application.

Devices, such as these present a practical difficulty: since flattened objects tend to align with a long axis along the beam axis, how does one keep a planar structure like this horizontal? The structure can be constrained mechanically. This can be achieved in at least three ways. Most simply, it can be trapped against a surface, either pushed or pulled against the surface [33]. While this is convenient for testing and demon-stration, it does not appear to be very useful for many practical applications. For practical deployment, mechanical restraint is possible by either enclosing it within a confined space, for example, between an upper and a lower surface, or mounting it on an axle of some type [56–60]. We consider this at the end of the chapter. These are not applicable to free-swimming micromachines.

For free-swimming rotors, two techniques have proved useful so far. First, the structure can be mounted on a stalk or axle fixed to the device [4,37,61–63]. This does not involve the difficulties in fabrication presented by moving parts, although it can necessitate a three-dimensional structure with undercuts that might be difficult or impossible to achieve using conventional photolithographic techniques for mass production. If the stalk is large enough, it will preferentially align with the beam axis, keeping the rest of the structure in the desired orientation. A disadvantage of the stalk can be that it makes it impossible to bring the main part of the device very close to an object situated above or below.

Second, if the structure is to be driven by an LG beam, the donut-shaped focal spot can be used to stably hold the structure in place [55]. Instead of a stalk, the structure can be provided with a hole, and if trapped in an LG beam with a focal spot matching the rotor in size, the beam can stably hold it horizontally. This relies on the

m0=±2

τ=QTP/w,

F=nQFP/C,

1093 Principles of design

hollow core of the LG beam, and is not suitable for trapping in Gaussian beams. An example of such a rotor is shown in Fig. 4.3.

3.4 NO ROTATIONAL SYMMETRY (p = 1)It is not necessary for a particle to possess discrete rotational symmetry to couple modes with different angular momenta—with p = 1, that is, no rotational symmetry, all possible angular momentum modes can be coupled (insofar as the size and other details of the structure allow). For example, irregular crystals, such as diamond chips will rotate in optical traps [64]. Such structures do not offer any particular advantage if driven by beams carrying orbital angular momentum (such as LG beams), so ap-pear to be best suited for driving by Gaussian beams. This requires the object to be chiral. A number of such devices have been demonstrated, and are typically helical or corkscrew-shaped [65,66]. This has the additional advantage of coupling rotational motion in the fluid to translational motion of the fluid—when rotated, they can func-tion as pumps [65]. It is possible to produce helical structures with discrete rotational symmetry—for example, a double helix will have p = 2, so one can combine both helical shape and discrete rotational symmetry [67].

3.5 USING REFLECTION TO GENERATE TORQUEFor the high-symmetry devices considered earlier, our governing design heuristic was the structure as a phase object transforming the orbital angular momentum. The

FIGURE 4.3 Optically Driven Micromachines Designed for Stable Trapping in a Laguerre-Gauss Beam.

Structures as shown in (A), (B), and (C) can be stably trapped using a LG beam with a focal spot matching the size of the structure. Since LG beams of opposite handedness can have the same focal spot size, this presents no difficulty for both-direction drive.

110 CHAPTER 4 Optically driven rotating micromachines

devices with p = 2 primarily operate by transforming the spin angular momentum of the light passing through. Thus, we have predominantly considered devices operat-ing on transformation of the transmitted light—devices based on refraction. How-ever, reflection also exerts forces on surfaces, and can be used for the design of micromachines [59,60,68].

The basic principles governing the relationship between rotational symmetry and coupling of angular momenta still apply. Corrugated or cross-shaped structures, such as considered earlier will generate torques through reflection. The reflection can be increased and made the dominant source of torque generation if the structure is suf-ficiently reflective. For example, they can be fabricated from high index materials, or produced with metallic coatings [59,60]. However, this will make them more dif-ficult, if not completely impossible, to trap and move with the driving beam, and highly reflective devices are best suited to fixed application. More challenging is to generate useful torque through reflection with free-swimming micromachines that can be moved and controlled using optical tweezers.

3.6 CONTROLLING REFLECTIONOne example of such a free-swimming reflection-based micromachine is an optically driven paddle wheel trapped and positioned using spherical handles [69]. An appli-cation of this could be to exert shear stresses on biological cells above or below the structure. Since the fluid flow due to an object rotating about a vertical axis is rela-tively small above and below it (and zero along the axis, if the object is symmetric), conventional refraction-driven micromachines that rotate about the beam axis are unsuitable. Here, we will consider further the optimization of a paddle wheel, such as reported in [69] and shown in Fig. 4.4. The paddle wheel is trapped by two tightly focused beams at the spherical handles, while a less focused beam is used to rotate the entire structure due to reflection forces on the paddles.

FIGURE 4.4 Optically Driven Paddle Wheel

This is a free-swimming optically driven micromachine which can be positioned three-dimensionally using two tightly focused beams to trap the spherical handles on the ends of the axle. Meanwhile, a third beam is used to drive rotation using reflection from the paddles.

1113 Principles of design

Ideally, we want the paddles to be as reflective as possible, and the handles to be as little reflective as possible. One approach to this could be to fabricate the paddle wheel from a very high index material, and use an antireflection coating on the han-dles. Alternatively, reflective coating could be used on the paddles. Either type of selective coating would be difficult to achieve in practice, so we will consider how the reflectivity can be controlled otherwise; we will consider antireflection coatings further later.

Noting that the reflectivity of spheres in optical traps is a function of their size as well as their refractive index, and the reflectivity varies approximately periodically with size [70], a size minimizing reflectivity can be chosen for the handles. For the paddles, there will be a similar behavior—when light reflected from the front and rear surfaces of the paddle interferes destructively or constructively, reflection will be minimized or maximized. Since we want the reflectivity to be maximized, we would choose a thickness of

λ+

n

2

1

4,

where n is a nonnegative integer, and λ is the wavelength of the light in the paddle. However, this is a simple ray optics or plane wave model, and the paddle would be driven by a focused beam. Therefore, it is useful to check the accuracy of this simple model. The paddle can be modeled using a hybrid T-matrix/DDA method [71], and the reflectivity determined for different thicknesses and different degrees of focused of the beam. This last parameter is important, since there will be competition be-tween gradient forces and reflection forces due to the driving beam, and if the gradi-ent forces exceed the reflection forces, no rotation will occur. On the other hand, if the beam is not focused tightly enough, not enough power will be delivered to the paddle, and reflection forces will be smaller.

For a paddle 4 µm in length, and 2 µm in width, of refractive index 1.6, illumi-nated by a beam with convergence corresponding to a numerical aperture of 0.8, the variation in total force (i.e., the sum of the gradient and reflection forces), and a func-tion of the paddle thickness and the position of the focal plane of the driving beam relative to the mid-plane of the paddle is shown in Fig. 4.5. The maximum force cor-responds to the most reflective paddle, with thickness of 3λ/4. Therefore, the simple ray/plane-wave model is usefully accurate. For this thickness of paddle, the depen-dence of the force on convergence of the beam is shown in Fig. 4.6A. As expected since tightly focused beams can trap spheres with refractive index of 1.6, the gradi-ent forces exceed the reflection forces for sufficiently tightly focused beam, and the paddle would not rotate. However, a continuously positive (i.e., pushing) force can be achieved with a lower convergence beam (including, e.g., a Bessel beam).

For a paddle of the optimum thickness determined here, but otherwise of the size in [69], the moment arm for the torque is 3.5 µm, the average force efficiency for a not-too-focused beam is approximately Q = 0.1 results in a force of 2 pN and a torque of 7 pN.µm for a pushing beam power of 6 mW, similar to results in [69]. This cor-responds to a torque efficiency of 0.33, which is reasonably high.

n2+14λ,

112 CHAPTER 4 Optically driven rotating micromachines

The importance of optimization of the reflectivity can be seen in Fig. 4.6B, which shows the force on a paddle of thickness λ/2. Due to the much lower reflectivity of a paddle of this thickness, the gradient force overcomes the reflection force, even for weakly focused beams, and the paddle is trapped rather than pushed.

We noted antireflection coatings earlier. These have been computationally and experimentally demonstrated in the past [72–74]. Their importance can be seen in Fig. 4.7 comparing axial trap strengths for typical (polystyrene, with refractive index n = 1.59) and very high index (titania, n = 2.48) particles; the titania particles are not trapped but are instead pushed axially out of the trap due to their high reflectivity.

This restricts the use of such high-index materials in micromachines where three-dimensional optical trapping is required. Considering the paddle wheel above, very high torque efficiency could be obtained by using titania paddles. As suggested ear-lier, one solution might be to produce a titania paddle wheel and coat the handles. The trapping forces on such core-shell particles, as shown in Fig. 4.8, can be readily calculated using generalized Lorenz-Mie theory applied to layered spheres [72,75].

The improvement in trapping is shown in Fig. 4.9. Where the titania particles could not be trapped previously, they can now be trapped for suitable choices of

FIGURE 4.5 Force Exerted on a Paddle by Reflection of a Beam with NA = 0.8, as a Function of Paddle Thickness and Distance of the Focal Point of the Beam from the Mid-Plane of the Paddle

It is important that the force remain positive for all positions of the paddle. Note that the peak forces correspond closely to a paddle thickness of 3λ/4. The force is given in terms of the force efficiency. The beam is propagating in the +z-direction.

1133 Principles of design

FIGURE 4.6

(A) Force exerted on a paddle of thickness 3λ/4, as a function of the position of the focus of the beam, and the convergence of the beam. The convergence of the beam is described by its numerical aperture; the force is given in terms of the force efficiency. (B) Force on a paddle of thickness λ/2, which is much less reflective. The gradient force overcomes the weaker reflection force, even for weakly focused beams, trapping the paddle.

114 CHAPTER 4 Optically driven rotating micromachines

FIGURE 4.8 The Geometry of a Coated Sphere Showing Radii and Refractive Indices

FIGURE 4.7 Axial Trap Strengths for Polystyrene (Refractive Index n = 1.59) and Titania (n = 2.48) Particles Trapped by a 1070 nm Beam with NA = 1.05

The axial trap strength is the force required to escape the trap in the axial direction in the direction of propagation of the beam. With our sign convention here, a negative trap strength indicates that the particle is trapped, and gives the maximum restoring force in the direction opposition to the beam propagation. Where the trap strength is positive, the particle is not trapped since the positive force still pushes the particle in the beam propagation direction. Unsurprisingly for such high index particles, the titania particles are not trapped.

1153 Principles of design

FIGURE 4.9

Improvement in axial trap strength for 1 µm radius (A) titania and (B) polystyrene particles, due to lower refractive index coatings, as a function of coating refractive index and thickness. The lines in (A) highlight the boundary between regions of trapping and no trapping.

116 CHAPTER 4 Optically driven rotating micromachines

coating thickness and refractive [and it should be noted that the 1 µm (core radius) particle considered here corresponds to a peak in Fig. 4.7, rather than an almost-trapped minimum]. The axial trap strengths achieved are close to those of uncoated polystyrene particles. Thus, axial trapping can be comfortably achieved, while the higher refractive index will allow enhanced radial forces [74].

Noting that micromachines can have more surfaces to reflect light than conven-tional microspheres, for example, due to internal structures, it is possible that some micromachines might be very difficult to trap and manipulate in three-dimensions. Thus, antireflection coatings might provide significant benefit even if lower index materials are used.

4 COMPUTATIONAL MODELINGAs can be deduced from our application of computational modeling based on exact theory (i.e., electromagnetic theory) in the previous section, such modelling can be a useful addition or replacement for the heuristic principles and approximate theories based on ray optics [7]. However, the complex geometries of many optically driven micromachines will force the application of general-purpose methods, which are usually much more computationally-demanding in terms of time and memory than special purpose methods designed for specific problems of geometries. The three most likely candidates as useful general purpose methods are the discrete dipole ap-proximation (DDA), the finite difference time domain method (FDTD), and the finite element method (FEM). In DDA, the object is divided into small volume elements, in which the effect of the incident field can be approximated by a uniform induced dipole moment (thus, it can be classified as a type of FEM). The discrete rotational and mirror symmetries seen in optically driven micromachines can be exploited to greatly speed up the calculations [71]. However, FDTD and FEM scale more gently with increasing size, so even if DDA is the method of choice for smaller microma-chines, sufficiently large ones can force the use of other methods.

What can be recommended? First, for sufficiently small and/or simple machines, fast methods, such as generalized Lorenz-Mie theory (GLMT) or the T-matrix meth-od [8,9] can be used. These can be combined with DDA to allow calculations for arbitrary shapes as long as they are not too large [71,76]. For larger problems, the best choice may well be the most familiar—if general purpose DDA [77], FDTD, or FEM codes are already in use, there seems to be no reason to not continue using it, as long as electromagnetic forces and torques can be calculated (which might not be possible with some codes without the availability of source code to modify).

5 FABRICATIONA common method of choice for fabrication of optically driven micromachines is two-photon photopolymerization (2PP) [78–80]. In this process, a UV-curing resin is polymerized using a two-photon process, where each photon has the energy of the

1176 Applications

UV photon; the light source is typically a short pulse near IR laser, commonly a fem-tosecond laser. Since the efficiency of the 2PP curing is proportional to the irradiance squared rather than the irradiance, three-dimensional structures can be produced without unintended polymerization above or below the focal spot. The volume cured by the focal spot without moving is a “voxel,” and voxel sizes of under 200 nm across and 500 nm long can be achieved without difficulty, allowing subwavelength resolu-tion of features. If higher resolution is required, techniques, such as using a second beam to restrict polymerization to a smaller region have achieved voxel widths of about 50 nm [79]. Very complex structures can be made. However, it is difficult to obtain a high degree of parallelization (some parallelization can be achieved by split-ting the femtosecond beam into multiple focal spots [67], and structures are often made one at a time. Thus, 2PP is very well-suited to development and prototyping, but may not be ideal for large scale manufacture.

The most successful large-scale manufacture used so far for optically driven mi-cromachines is the self-assembly of vaterite microspheres [38–40], as described ear-lier. If more complex shapes can be readily and cheaply self-assembled, this could prove similarly useful, and may prove a productive line of research.

Otherwise, the design of optically driven micromachines that are suited for con-ventional large-scale photolithographic techniques (even if prototypes are made with 2PP) could prove the gateway to widespread deployment.

6 APPLICATIONSMuch of the research on optically driven micromachines has focused on investiga-tion of the basic physics of optical angular momentum, or proof-of-principle dem-onstrations of designs and fabrication. However, they are also used for research on a broader range of topics, as tools rather than as objects of research. Just as optical tweezers are widely used as tools for the measurement and application of forces, optically driven rotating micromachines can be used as tools for the measurement and application of torques. Two promising fields are microrheology, where rotation offers higher spatial resolution due to less effect from nearby surfaces [38,81–84], and the application of torques or forces to biomolecules and cells, either directly by the micromachine [41] or indirectly via fluid flow [85].

Prominent among more traditionally “machine-like” applications are those in-volving interaction with fluids, such as pumps, mixers, and flowmeters [60,65,86]. This is the common feature of almost all work on and with optically driven micro-machines—they are intended to function in a liquid environment. Breaking with this trend is a recent body of work including rheology of gaseous media [87] and rotation in a vacuum, with the achievement of rotation rates as high as 5 MHz [88,89].

Widespread use is likely to come from applications, such as the pumps, mixers, and flowmeters noted earlier, deployed in microfluidic devices, and intended for use in laboratories not specialized in optics. Many devices developed so far are intended for such applications. Finally, we present a case study of the development of such a device.

118 CHAPTER 4 Optically driven rotating micromachines

6.1 A CASE-STUDY: PINNED CROSS-ROTORSOne possible application of optically driven micromachine is to measure or monitor changes in the viscosity in a microfluidic device. A microfluidic device can be used to determine the viscosity: the flow rate can be set in by a pump, and then the viscos-ity found (it is also useful to know the ambient temperature). However, this gives the average viscosity throughout the entire device. If there is a reaction chamber where the local chemical composition or the temperature is unknown, the local viscosity is unknown, and measurement can be useful. An optically driven micromachine can be a suitable probe to determine the local properties of the fluid flowing in the microflu-idic device. In order to prevent the machine from being washed away by the flow, it can be mounted on an axle about which it can rotate. Here, we present a case-study of the development of such a device. Such devices are not simply achieved and some-times need redesign from the concept to production stages.

The initial design of cross rotor was originally conceived of as a single step pro-cess where a base and rotor would be produced together, shown in Fig. 4.10. An end cap could prevent the rotor being lost in the fluid and allow the device to operate in any orientation. The devices were made using 2PP on a commercial system (Nano-Scribe). The assumption which meant that we tried this process is that given a small enough attachment area the rotor could be dislodged from any superficial surface attachment with a microactuator (such as a mechanically controlled micropipette). This design was partially successful but the microactuator step would not produce a freely rotating rotor with a high repeatability. Most times that this was attempted, the rotor would rotate initially, but would become trapped in an orientation where it could not be used or the stalk bent by the large forces from the micropipette.

FIGURE 4.10 Initial Design and Production of Pinned Cross-Rotors

(A) Shows the design, (B) the cross-rotors being made using two-photon photopolymerization, and (C) an SEM image of a completed rotor. The axle has a base for secure attachment to the substrate, and a cap to stop the rotor from escaping. Rotors are made with a gap between them and the axle and base, to allow for free movement. This last part of the design was unsuccessful since the rotors would usually not rotate freely.

1196 Applications

The solution was a different design, consisting of two manufacturing steps, whereby the base-axle was made using 2PP as before, but without the rotors. The rotors were then made separately. This required the freeing of the rotors from the glass substrate. This is most simply done by photopolymerizing the structures onto a sacrificial layer, rather than directly onto the substrate; an example of this is shown in Fig. 4.11. The properties of the sacrificial layer are dictated by the solvent used to wash away uncured polymer. In this case, polyvinyl alcohol (PVA) was chosen, since it is soluble in water but not in the isopropanol used to wash the unpolymerized photopolymer away. After washing, the rotors were pipetted onto the substrate on which the bases had been made. Holographic optical tweezers were used in a four-beam configuration to grab the arms of the rotor, and move them over the axles and thread them on. After threading, the optical tweezers were converted from the four-beam configuration into a Laguerre-Gauss beam to drive the rotors using the transfer of orbital angular momentum.

Further improvements to this design are possible, such as twisting the arms of the cross rotor as shown in Fig. 4.12. This can achieve three optimizations. First, the contact area and friction between the rotor and the base can be reduced. Second, the torque efficiency for a particular handedness of driving beam can be improved. Third, a chiral object of this type with the appropriate shape can gener-ate a force lifting it as it rotates, lifting the rotor from the base and reducing friction and viscous drag.

Possible applications for a micromachine, such as this in a microfluidic device for the measurement of viscosity have already been mentioned. Another plausible ap-plication is as a flowmeter. Rotation of a rotor next to a wall, as shown in Fig. 4.13, would allow flow rates to be measured. The rotor would not require optical driving for such an application (after assembly), but laser illumination could be still useful

FIGURE 4.11 Use of a Sacrificial Layer to Free Structure Created Using 2PP

The substrate was coated with polyvinyl alcohol (PVA), and spheres produced on both the coated and uncoated regions. The unpolymerized resin was washed off using isopropanol, after which spheres were present on both the coated and uncoated areas. After washing with water, the spheres on the coated area have been freed.

120 CHAPTER 4 Optically driven rotating micromachines

FIGURE 4.12 Improved Rotor With Twisted Arms

This introduction of chirality will allow higher torque efficiencies for one handedness of driving beam, and can be used to provide hydrodynamic forces to lift the rotor from the base for reduced drag.

FIGURE 4.13 A Potential Application of the Fabricated Rotor Structure: A Flowmeter

The fluid boundary conditions dictate that a differential flow rate near the wall will cause a freely moving rotor to spin. Optical readout or video microscopy could be used to determine the flow from the rotation rate.

1216 Applications

for measurement of the rotation rate. However, if a flow will drive rotational motion, we can use rotational motion to drive a flow, and the machine could be used as a pump, similarly to those discussed earlier. A cross-rotor pump, such as this can be driven using a static diffractive optical element to convert an incident Gaussian beam into a beam carrying orbital angular momentum [61].

The viscosity of a liquid can be determined by either passive or active measure-ments. Passive measurements entail observation of the behavior of an object without actively driving its motion. Passive measurements can be performed by looking at the Brownian motion in a free fluid or under the influence of a static external potential. For a micromachine that is angularly trapped, such as a vaterite microsphere trapped by a linearly polarized beam, this can be done [84]. However, while a micromachine, such as this pinned rotor can be angularly trapped using, for example, four beams, with one on each arm (in much the same way as it was assembled), the Brownian motion needs to overcome friction as well as viscous drag. While a very small force will drive (slow) motion against viscous drag, friction is a different matter (as noted by Aristotle millennia ago [90]), and it is likely that Brownian motion will have no effect. Therefore, active measurements are required for viscosity measurements in the presence of friction.

Since the viscous drag constant is unknown a priori for a pinned rotor, due to enhancement of drag by the proximity of the base as well as the complex shape, cali-bration measurements to determine it are useful. It is not necessary to measure the op-tical torque driving the motion, since that can be included in the calibration constant. Essentially, if the rotor is rotated in a fluid of known viscosity η at a known angular speed Ω by an unknown torque τ, we can relate these by a drag constant D such that

τ η= ΩD .

Thus, the ratio τ /D can be determined, and sufficient to measure the viscosity in other fluids as long as the same beam and the same beam power are used. If the beam power is changed, but the structure of the beam remains the same, the linearity of the optical torque with beam power allows the viscosity to still be found.

This requires measurement of the rotation rate, or equivalently the angular posi-tion of the rotor over time. A cross-rotor can be tracked using video microscopy and digital signal analysis. The outline of a rotor shaped object can be found using a gra-dient of intensity method. The angular position can be determined by matching the object features with a graphical template equivalent. The graphical template, having explicit angular information can be made to strongly overlap with the observed pat-tern. Thus the angle of the object is determined by reading off the angle required for the matching template. An alternative is to locate the edge positions and orientations with subpixel accuracy using an algorithm based on the partial area effect [91]. This algorithm shows high accuracy for both simulated and real images. Tests on simulat-ed noisy (standard deviation 20%) images show that root mean square error (RMSE) of the cosine angle of the edge is approximately 0.05. With appropriate smoothing and filtering, one can obtain the angular position from the real images, with RMSE of 0.04. A test with a digital camera gives an averaged error of 2.7 degree.

τ=DηΩ.

122 CHAPTER 4 Optically driven rotating micromachines

In Fig. 4.14, we show the measured rotation rate of the rotor. The rotation rate was determined from video frames, such as those shown in Fig. 4.15, using a MATLAB code derived from the subpixel edge detection algorithm in [91]. The rotation rate was measured for rotation by LG beams of differing azimuthal mode index m varying from m = 6 to m = 20. The highest rotation rates occurred for a driving beam with m = 13.

FIGURE 4.14 Measurement of the Rotation of a Rotor

(A) Measurement of rotation over time, using detection of angle. (B) Rotation rate as a function of m, the azimuthal mode index of the driving LG beam.

123 References

7 CONCLUSIONSWe have presented the basic theory governing the design and operation of optically driven micromachines. The close relationship between symmetry and optical torque provides some simple design principles that can be used to develop optically driven micromachines. Simple approximate theories based on ray optics and plane waves can also usefully guide development. Simple heuristic principles of these types are often in close agreement with exact computational modeling and experimental test-ing, but are much simpler and quicker to apply. Finally, we surveyed some appli-cations, and presented a case study of the development of a prototype for such an application: a cross-rotor pinned to a surface, for use in microfluidic devices as a viscometer.

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[89] Arita Y, Mazilu M, Vettenburg T, Wright EM, Dholakia K. Rotation of two trapped mic-roparticles in vacuum: observation of optically mediated parametric resonances. Opt Lett 2015;40:4751–4.

[90] Aidun J. Aristotelian force as Newtonian power. Phil Sci 1982;49(2):228–35. [91] Trujillo-Pino A, Krissian K, Alemán-Flores M, Santana-Cedrés D. Accurate subpixel

edge location based on partial area effect. Image Vision Comput 2013;31(1):72–90.

129

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00005-7Copyright © 2017 Elsevier Ltd. All rights reserved.

Jianhe Guo*, Donglei (Emma) Fan*,***Materials Science and Engineering Program, The University of Texas at Austin, Austin, TX,

United States; **The University of Texas at Austin, Austin, TX, United States

CHAPTER OUTLINE

1 Introduction ...................................................................................................... 1292 Optically Enabled Robotic Micro/Nanodevices .................................................... 130

2.1 Nanomanipulation and Robotization With Optical Traps .....................1312.2 Nanomanipulation and Robotization With Controlled

Photon Momentum.........................................................................1373 Plasmonically Enabled Robotic Micro/Nanodevices ............................................ 1454 Electrically and Optoelectronically Enabled Robotic Micro/Nanodevices .............. 150

4.1 Electric Tweezers Enabled Robotic Micro/Nanodevices .......................1504.2 Optoelectronic Tweezers Enabled Robotic Micro/Nanodevices .............155

References .............................................................................................................. 158

1 INTRODUCTIONSince Richard Feynman raised the concept of miniaturized machines in his ground-breaking talk “There’s plenty of room at the bottom” in the 1950s [1], micro/ nanoscale machines/robotic devices have attracted immense interest in research. In the last few decades, with the vigorous progress in nanoscience and technology, micro/nanoscale materials can be fabricated with designed dimensions, geometries and chemistry that exhibit unique electric, magnetic, mechanical, and optical properties, owing to large surface areas and quantum confinement effects. Recently, by exploiting and con-trolling the interactions of electromagnetic waves with micro/nanoscale materials, remarkable advances have been made in the realization of micro/nanoscale robotic devices by using nanoentities as building blocks. In this chapter, we provide a broad

Electromagnetic wave enabled micro/nanorobotic devices and their applications

5

130 CHAPTER 5 Electromagnetic wave

survey of the recent progresses in the development of electromagnetic wave enabled micro/nano mechanical motors. The topics are categorized according to the em-ployed driving mechanisms. The focus is on the fundamentals, device performance, and applications. The aim is to provide readers a sense of the many promising pro-gresses so far, which support the foundations of light robotics. Where appropriate, readers are referred to the other chapters in this book that provide details, updated results and applications of the various topics that are reviewed here. Light robotics can benefit from recent emerging techniques beyond the traditional optical trapping and manipulation of dielectric particles and, hence, we expand our scope and include other electromagnetic wave-enabled approaches for the micro/nano domain, such as plasmonics, optoelectronic tweezers, and even electric tweezers.

2 OPTICALLY ENABLED ROBOTIC MICRO/NANODEVICESThe most well-known optical technique for manipulating micro/nanorobotic devices are the so called “optical tweezers,” which are also referred to as “single-beam gra-dient force traps.” The manipulation is based on forces (typically on the order of piconewtons) exerted by a highly focused laser beam, which can trap and transport micro/nanoscale objects. The involved optical forces can be categorized into two types: scattering optical forces and gradient optical forces. A proper fundamental understanding of the optical forces depends on the size of the trapped objects relative to the wavelength of the employed lasers as shown in Fig. 5.1 [2]. In the Mie scat-tering regime, where the objects are much larger than the wavelength of laser beam, scattering optical forces dominate the trapping effect. The refraction and reflection of light by the trapped objects cause redirection of photon momentum. According to Newton’s third law, the objects will also receive a backward force of magnitude equal to the rate of change of optical momentum. As a result, the refraction light induces the attraction of the objects to the region of highest light intensity and the reflected light compels the objects along the direction of laser beam propagation (Fig. 5.1A–C). For particles smaller than the wavelength of light, where Rayleigh scattering dominates, the scattering forces can be determined by the Fresnel equa-tions and electromagnetic wave optics. The propulsion force Fscat along the axis of the laser beam is given by [3]:

πλ

= −+

FI

c

r m

mn

128

3

1

2,bscat

05 6

4

2

2

2

(5.1)

where I0 is the intensity of laser beam, r is the radius of the trapped particle, m is ratio of the refraction index of the particles and the medium, λ is the wavelength in vac-uum and nb is the refraction index of the medium. In the Rayleigh scattering mode, the trapping of objects is mainly due to the gradient of the optical forces. As shown in Fig. 5.1D, the objects are electrically polarized by the electric component of the optical field. The interaction of the polarized objects with the electric component of the optical field results in the transport of the object along the gradient to the highest

Fscat=I0c128π5r63λ4m2−1m2+22nb,

1312 Optically enabled robotic micro/nanodevices

intensity of the optical field. The gradient force (Fgrad) for a spherical particle due to Rayleigh scattering is given by [3]:

= − −+

∇Fn r m

mE

2

1

2,b

grad

3 3 2

22

(5.2)

where E is the electric field of the laser beam.

2.1 NANOMANIPULATION AND ROBOTIZATION WITH OPTICAL TRAPSAlthough optical traps created by focused laser beams are generally considered as a static trapping technique, the control of physical motion of the optical traps via preci-sion stages has been widely used in manipulating various micro/nanoparticles. Since

Fgrad=−nb3r32m2−1m2+2∇E2,

FIGURE 5.1 Schematic Diagrams of Manipulation Mechanisms of Optical Tweezers

(A,B) For particles of sizes in the Mie scattering regime, the refraction of nonuniform light by the particles generates a net force along the optical field gradient toward the highest optical intensity. (C) The reflection of light results in a radiation pressure along the laser beam axis. (D) For particles with Rayleigh scattering, the electromagnetic force on light-induced electric dipoles in the particles compels the particles to transport along the gradient toward the highest electric field.Reproduced with permission Dholakia K, et al. Optical micromanipulation. Chem Soc Rev 2008;37(1):42–55

[2]. Copyright (2008) Royal Society of Chemistry.

132 CHAPTER 5 Electromagnetic wave

Arthur Ashkin observed the trapping of dielectric microparticles by a focused laser beam for the first time in 1986 [3], dielectric microparticles have become extremely desirable as microhandles in manipulating and applying forces to biological systems.

In a simple design, single beam optical tweezers are applied to trap just one mi-cro/nanobead, which links to interesting biomolecules as shown in Fig. 5.2A. Given the fixed position of the optical trap, it can be known that there is a tensional force applied from the biomolecules to the micro/nanobead that result in its displacement. In a range up to several 100 nm, the tensional force on the micro/nano bead is a linear function of displacement, x, from equilibrium given by:

= −F k x ,trap bead (5.3)

where ktrap is the trap stiffness. This simple design has been widely used in the in-vestigation of various processive molecular motors, which can move continuously along chain structures attached to the substrate, such as conventional kinesin and RNA polymerase [4–6]. Examples of nonprocessive molecular motors will be dis-cussed later in comparison. Here, in the manipulation of processive molecule mo-tors, the force applied increases as the protein displaces the bead from the trapping center until the molecular motor reaches the maximum force and stalls. In a more

F=−ktrapxbead,

FIGURE 5.2 Optical Tweezers System Configurations for Single-Biomolecule Investigation

Single beam optical tweezers: (A) single trap design and (B) dynamic force spectroscopy. Double trap optical tweezers: (C) isometric clamps and (D) three-bead assays.

Reproduced with permission Capitanio M, Pavone FS. Interrogating biology with force: single molecule

high-resolution measurements with optical tweezers. Biophys J 2013;105(6):1293–1303 [14].

Copyright (2013) Elsevier.

1332 Optically enabled robotic micro/nanodevices

sophisticated dynamic design, also referred as “isotonic clamps,” with a position detector based feedback system, the optical trap actively displaces, following the movement of the micro/nanobead, to keep both the displacement (x) and thus the tension force constant. In this configuration, the isotonic condition allows a direct observation of the force-dependent conformational transition process [7,8]. The bio-molecule movements are also accurately determined in real time, which enables the measurement of the mechanical output of the molecular motors. Chapter 3 presents an alternative approach, shape-induced forces, which uses designed shapes to main-tain the optical force even when the object moves relative to the trap. Another dy-namic strategy is the so called dynamic force spectroscopy (DFS) [9]. It determines

the rupture forces of molecular bonds at a constant loading rate

dF

dt as shown in

Fig. 5.2B. The constant loading rates are achieved by dragging the trapped particles

at a constant speed

dx

dtbead or increasing the laser power at a constant rate

dk

dttrap

[10,11]. This strategy is commonly used for measuring weak bonds, of which the

reaction kinetics is too high for isotonic clamps to maintain constant distance. Mo-torized mirrors are often applied in these dynamic optical beam controls to steer the laser beam and move the trap. Acousto-optic deflectors or electro-optic deflectors [12,13] can achieve response with a much higher speed for this purpose.

Double optical tweezers that trap two micro/nanobeads simultaneously enable more sophisticated manipulation. This configuration has been widely employed to investigate mechanical properties of various biomolecules including double strand-ed DNA, single stranded DNA, and single RNA molecules [15,16]. As shown in Fig. 5.2C, each end of the individual molecule is attached to one of the trapped beads, where one bead is trapped at a fixed position and the other is displaced to stretch the molecule. This technique, also called “isometric clamps,” was recently used to investigate the folding mechanisms of single protein molecules [17]. Three-bead assays are also designed to study nonprocessive molecular motors, of which the bonding dissociation occurs in just one step instead of a series of steps as that of processive motors. For instance, as shown in Fig. 5.2D, in the investigation of the interaction between skeletal muscle myosin and actin, two optically trapped beads attach to and suspend an actin filament. A third bead binding the myosin molecule reports its position [18], while the conformational shift and force in the interaction of myosin and actin are measured by the two optical traps.

The applications of these robotic micro/nano dielectric particles are not limited to the investigation of single biomolecules. As shown in Fig. 5.3A, this technique has also been applied to study the mechanical deformation of human red blood cells under tensile forces of hundreds of picoNewtons [19]. With the development of mul-tiple optical traps, several micro/nanobeads can be captured simultaneously and ar-ranged into various functional devices [20–22]. A micropump can be assembled by several silica microspheres as shown in Fig. 5.3B [20]. The “snakelike” motions of this pump is realized by moving the optical traps in a propagating sine wave fashion,

dFdt

dxbeaddtdktrapdt

134 CHAPTER 5 Electromagnetic wave

FIGURE 5.3 Manipulation of Various Microbeads-Based Devices by Moving Optical Traps

(A) Schematic and optical images of the deformation of human red blood cells by microbeads manipulated by optical traps. (B) Schematic of an undulatory microfluidic pump using flexible microbead assembly and snapshots showing the movement of a tracer particle due to pumping of the assembled beads. (C) Optical snapshots showing the operation of a light-switched valve in a particle sorting microchannel.

Part A: Reproduced with permission from Lim CT, Dao M, Suresh S, Sow CH, Chew KT. Large deformation

of living cells using laser traps. Acta Materialia. 2004;52(7):1837–1845 [19]. Copyright (2004) Elsevier;

Part C: Reproduced with permission from Terray A, Oakey J, Marr DWM. Microfluidic control using colloidal

devices. Science 2002;296(5574):1841–1844 [20]. Copyright (2002) American Association for the

Advancement of Science.

1352 Optically enabled robotic micro/nanodevices

which agitates flows in a microchannel. With a similar strategy, microvalves made of assembled microspheres can be operated by optical traps in the T-bifurcation of a microchannel (Fig. 5.3C) [22]. Unlike the “snakelike” micropump, the microspheres here are rigidly attached during assembly by laser-initiated photopolymerization in a photosensitive polymer solution. When in operation, one end of the device is fixed next to a channel wall by an optical trap, and the body is rotated by another optical trap to direct flows of microparticles (Fig. 5.3C). Chapter 2 discusses several optical microassembly techniques.

With significant advances in the fabrication and characterization of nanowires, the practical use of nanowires as active components of devices is a pressing issue. It is of dire need to develop new tools to manipulate and integrate nanowires with high precision and efficiency. Grier’s group in 2005 demonstrated the trapping, aligning, translating, and rotating of an individual semiconductor nanowire by an array of opti-cal traps as shown in Fig. 5.4A. Here, a nanowire is translated either by moving the optical trap array or by moving the sample stage relative to the optical trap array [23]. The trapping and transport of various semiconductor nanowires in a single-beam optical trap were later realized as shown in Fig. 5.4B [24]. The manipulation of these nanowires enables the assembling and construction of designed complex structures as shown in Fig. 5.4C. Furthermore, a nanowire can be accurately maneuvered to

FIGURE 5.4 Manipulation of Nanowires by Moving Optical Traps

(A) Schematic and optical images of a single semiconductor nanowire manipulated by multiple optical traps. (B) Schematic of the manipulation process of a semiconductor nanowire by a single optical trap. (C) Schematic and optical dark-field image of light-assembled nanowires. (D) Schematic and optical dark-field image of a nanowire scribing the surface of a cell using optical tweezers.

Part A: Reproduced with permission from Agarwal R, Ladavac K, Roichman Y, Yu G, Lieber CM, Grier DG.

Manipulation and assembly of nanowires with holographic optical traps. Opt Express. 2005;13(22):8906–

8912 [23]. Copyright (2005) Optical Society of America; Part D: Reproduced with permission from

Pauzauskie PJ, Radenovic A, Trepagnier E, Shroff H, Yang P, Liphardt J. Optical trapping and integration of

semiconductor nanowire assemblies in water. Nat Mater 2006;5(2):97–101 [24]. Copyright (2006) Nature

Publishing Group.

136 CHAPTER 5 Electromagnetic wave

scribe over the membrane of a live cell as shown in Fig. 5.4D. The cell remains alive after the process, where an infrared laser is selected to minimize cell damage. These results suggest the feasibility of using nanowires as mechanical stimuli and drug delivery vehicles for single-cell study. Moreover, owing to the unique optical properties of semiconductor nanowires, they have also been demonstrated as wave-guides for light illumination inside live cells. Chapter 8 discusses the optimization of lithium niobate nanowires for waveguiding and second harmonic generation.

With the significant advances of nanofabrication techniques, including electron beam lithography and two-photon polymerization, various complex micro/nano-structures have been produced and operated as useful nanodevices by the dynamic optical traps. As shown in Fig. 5.5A, novel force probes can be fabricated by two

FIGURE 5.5 Sophisticated Microstructures Driven by Optical Traps

(A) Optical images and schematic of various microprobes manipulated by multiple optical traps. (B) Schematic, scanning electron microscopy (SEM), and optical images of a micropump driven by circularly scanning dual optical traps; (C) Schematic, SEM, and optical images of a microdisk pump driven by circularly scanning three-point optical traps (overlay: trajectory of a tracer particle from snap shots taken every 2 s).

Part A: Reproduced with permission from Phillips DB, Gibson GM, Bowman R, Padgett MJ, Hanna S,

Carberry DM, et al. An optically actuated surface scanning probe. Opt Express 2012;20(28):29679–29693

[25]. Copyright (2012) Optical Society of America; Part B: Reproduced with permission from Maruo S, Inoue

H. Optically driven micropump produced by three-dimensional two-photon microfabrication. Appl Phys Lett

2006;89(14):144101 [26]. Copyright (2006) AIP Publishing LLC; Part C: Reproduced with permission

from Maruo S, Inoue H. Optically driven viscous micropump using a rotating microdisk. Appl Phys Lett

2007;91(8):084101 [27]. Copyright (2007) AIP Publishing LLC.

1372 Optically enabled robotic micro/nanodevices

photon polymerization for measuring surface topography [25,28,29]. The nanoprobe handles are designed with a cylindrical shape. Transverse to the long direction, the whole handle is strongly confined by multiple optical traps; along the long direction, which is also the sensing direction, the trapping force is much lower compared to that in the transverse direction. Loaded with a nanoscale tip, these optically enabled probes have been demonstrated to provide a spatial resolution of 11 nm along the sensing axis and a force sensitivity of less than 150 fN (femtoNewton) given the Brownian motion calibration. Chapter 3 discusses the theoretical and experimental details of surface topography applications while Chapter 12 discusses applications in single-molecule force sensing.

The optical tweezers technique not only can be used to linearly transport micro/nanostructures but also to rotate them. One way to rotate microstructures is to ap-ply a laser beam with a rotating nonuniform intensity profile [30–32]. Optical traps can rotate a nanorod inside a Elodea densa plant cell up to 4 Hz [32]. The viscosity inside the cells can be monitored by analyzing the intracellular rotation speed of the nanorod. A micropump consisting of two lobed rotors integrated in a microchannel and driven by circularly scanning dual optical traps was later studied by Inoue et al. as shown in Fig. 5.5B [26]. The lobed rotors are confined by their own shafts at fixed locations, although the optical traps are not focused at the center of the rotors. The velocity of microflows is proportional to the rotation speed of the rotors in the range of 0.2–0.7 µm/s. Extending the concept to a circularly scanning three-point optical trap, a microdisk pump is developed as shown in Fig. 5.5C [27]. Here a microrotor is trapped and driven by the rotation of an optical trap in a U-shaped microchan-nel. The average flow speed reaches 1 µm/s when the speed of the micropump is at 30 rpm. Although the demonstrated flow speed is not high enough for practical applications, with optimized design, the micropumps could produce sufficient flow speed and make great impact on various lab-on-a-chip devices.

2.2 NANOMANIPULATION AND ROBOTIZATION WITH CONTROLLED PHOTON MOMENTUM2.2.1 Nanomanipulation of asymmetric structures by controlling linear photon momentumMicro/nano entities are mechanically manipulated by their interaction with the opti-cal momentum carried by an incident light. Optical momentum has both linear and angular components. As aforediscussed (Fig. 5.1A), a symmetric microobject can receive a net transverse force driving it toward the axis of the laser beam. When positioned on the central axis of the laser beam, there is no net transverse forces or torques on the object anymore. However, the torque can be nonzero if the microob-ject has asymmetric geometry. Chapter 4 discusses basic principles and heuristics for designing light-driven rotating microstructures.

Higurashi and coworkers reported the first design of rotatory micromotor driven by radiation pressure in 1994 [33]. As shown in Fig. 5.6A, an optical torque is gen-erated on the asymmetric objects even when there is no net force applied by the static illumination. In this work, the rotation speed of the rotary motor made of silica

138 CHAPTER 5 Electromagnetic wave

linearly increases with laser power and can reach a speed greater than 20 rpm at 80 mW. In general, optically induced torques require a structural design with rotational symmetry but not bilateral symmetry. The rotation is usually along one direction only, either clockwise or counterclockwise, which is determined by the designed geometry. By using advanced nanofabrication techniques, including electric beam lithography and two-photon polymerization, various sophisticated microscopic ma-chines haven been produced and successfully driven by linear-photon resulted ra-diation pressure as shown in Fig. 5.6B–C [34,35]. For instance, a micromachine is designed consisting of a central rod and a sprinkler structure with eightfold rotational

FIGURE 5.6 Rotation of Micromachines Using Anisotropic Structures and Linear Optical Momentum

(A) The principle of torque generation on a micromotor with an anisotropic geometry. (B) SEM images of two types of microrotors fabricated by electric beam lithography. (C) Schematics and optical images of a micromachine fabricated by two-photon polymerization in a rotation mode (bottom) and in a static mode (top).

Part B: Reproduced with permission Higurashi E, Ohguchi O, Tamamura T, Ukita H, Sawada R. Optically

induced rotation of dissymmetrically shaped fluorinated polyimide micro-objects in optical traps. J Appl

Phys 1997;82(6):2773–2779 [34]. Copyright (1997) AIP Publishing LLC; Part C: Reproduced with

permission Galajda P, Ormos P. Complex micromachines produced and driven by light. Appl Phys Lett

2001;78(2):249–251 [35]. Copyright (2001) AIP Publishing LLC.

1392 Optically enabled robotic micro/nanodevices

symmetry, which aims to increase its stability and to obtain efficient and uniform rotation performance, respectively (Fig. 5.6C) [35].

By strategically adjusting the focus points of a laser beam, a microrotor with dual rotation orientations has been achieved by Ormos and Galajda in 2002 [36]. The em-ployed strategy is illustrated in Fig. 5.7A. First, a spiral structure is designed to serve as the rotor (Fig. 5.7B), to which the torque transfer due to the reflection of linear opti-cal momentum can be maximized. Then, in an inverted microscope setup, by shifting the relative positions of the focus point of the laser and the center position of the spiral motor in the vertical direction, the rotation direction of the microrotor can be readily altered due to the direction change of incident photons (Fig. 5.7C). Both simulation and experimental results of the dependence of rotation speed and direction on the axi-al position of the objective have been reported. This work presented a straightforward approach to switch the rotation orientations of micromachines. In Chapter 6, Ormos et al. presented a variety of tools that they have developed over the years.

Based on the redirection of linear photon momentum by the simple optical twee-zers, the aforediscussed micromotors have demonstrated excellent performances.

FIGURE 5.7 A Microrotor With Dual Rotation Orientations Compelled by Linear Photon Momentum and Radiation Pressure

(A) Schematics of change of rotation orientation depending on the relative positions of the rotor and the focus point of laser. (B) Schematic and optical image of the microrotor. (C) Experimental (solid line) and simulation (dashed line) results of rotation speed of the micromotor versus its vertical position.

Reproduced with permission from Galajda P, Ormos P. Rotors produced and driven in laser tweezers with

reversed direction of rotation. Appl Phys Lett 2002;80(24):4653–4655 [36]. Copyright (2002) AIP

Publishing LLC.

140 CHAPTER 5 Electromagnetic wave

They can be trapped stably during rotation and regulate ultra-low flow rates pre-cisely. Recently, more complex radiation-pressure driven micromotors have been developed in the same principle and applied in microfluidics for generating and con-trolling flows [37,38]. As shown in Fig. 5.8 [38], a micropump is designed based on a microrotor with twin spiral blades positioned on a single rotational axis. The two blades have opposite spiral chirality (Fig. 5.8A–B). When an incident beam focuses on the axis between the twin spiral rotors, the optical radiation pressure generates optical torques on both spiral blades in the same direction. As a result, the total optical torques applied on the twin spiral microrotor doubles that of a single spiral

FIGURE 5.8 Micropump With a Twin Spiral Microrotor Design for Faster Rotation Driven by Linear Photon Momentum

(A) Schematics of the micropump and the twin spiral microrotor. (B) SEM image of the microrotor. (C) Transport of a tracer particle in microfluidic channel by the twin spiral microrotor.Reproduced with permission from Maruo S, Takaura A, Saito Y. Optically driven micropump with a twin spiral

microrotor. Opt Express 2009;17(21):18525–18532 [38]. Copyright (2009) Optical Society of America.

1412 Optically enabled robotic micro/nanodevices

microrotor. When confined to a U-shaped microchannel by a stable optical trap, the twin spiral microrotor can rotate to 560 rpm at 500 mW. A tracer particle can be pumped through the channel when the twin spiral microrotor rotates at 300 rpm. The flow rate is estimated as 18 pL/min.

A waterwheel micromachine driven by radiation pressure has also been devel-oped [39,40]. As shown in Fig. 5.9, a waterwheel micromachine made of multiple in-tegrated components is fabricated by using the two photon polymerization technique. It consists of a cogwheel rotor attached to an axle bearing with an optical waveguide positioned in the tangential direction of the rotor. The operation of the device relies much less on bulky optical instruments compared to other micromachines operated by the same mechanism. For instance, the laser excitation is introduced by an optical waveguide avoiding the use of lens and objectives. This type of microrotors can be rotated by an optical power as low as 5–10 mW and reach a speed higher than 10 Hz at 300 mW. It inspires a new research effort in integrating all components of opti-cally driven micromachines on a single chip.

2.2.2 Nanomanipulation by controlling angular photon momentumAlthough not as straightforward as the use of forces and torques generated by linear optical momentum, micro/nanoobjects can also be manipulated by controlling angu-lar photon momentum. Optical manipulation based on angular photon momentum is closely linked to the discovery of angular momentum of light. During the first attempt of measuring spin angular momentum of circularly polarized light by Beth in 1936, a birefringent plate was rotated due to the counter force generated by the change of the polarization of light passing through the plate [41]. Angular momentum consists of a spin component and an orbital component. The former is associated with optical field gradient and polarization state of light [41,42]. A spin angular momentum of ħ is assigned to each photon of circularly polarized light. Orbital angular momentum, on the other hand, is not associated with polarization but, rather, depends on spatial

FIGURE 5.9 Integrated Illumination of Paddlewheels Driven by Linear Optical Momentum

(A) Schematic, (B) optical, and (C) SEM images of the system.Reproduced with permission from Metzger NK, Mazilu M, Kelemen L, Ormos P, Dholakia K. Observation and

simulation of an optically driven micromotor. J Optics 2011;13(4):044018 and Kelemen L, Valkai S, Ormos

P. Integrated optical motor. Appl Opt 2006;45(12):2777–2780 [39,40]. Copyright (2011) IOP Publishing

Ltd and copyright (2006) Optical Society of America.

142 CHAPTER 5 Electromagnetic wave

distribution of the field. Orbital angular momentum (OAM) is quantized as lħ per photon, where the integer l is the so called topological charge of singularity. Orbital momentum can exceed spin angular momentum depending on the value of l. The transfer of both forms of angular momenta to a trapped micro/nanoobject through birefringence or absorption can compel micro/nanoobjects into circular motion.

Based on transfer of spin angular momentum, a trapped calcite crystal can be rotated at a speed over 350 Hz by Friese and coworkers in 1995 [45]. The rotation torque per unit area on the trapped birefringent particle is given by:

τ εω ( )( )( )= ± − −c

E kd n n2

1 cos ,02

0 e (5.4)

where c is speed of light, ε is the permittivity of the medium, w is the optical fre-quency, E0 is the electric field strength, k is the wave number of the incident light, d is the thickness of the material, and n0 and ne are the ordinary and extraordinary refractive indices of the material, respectively. Note that the optical induced torque is proportional to square of electric field, and thus it linearly increases with light intensity. The birefringence strategy allows achieving high rotation speeds with high power laser illumination without side heating effects using transparent birefringent materials. Furthermore, driving micro/nanoparticles to rotate is not only essential for the fundamental understanding of angular momentum of light: it also provides a unique approach to determine viscous drag and viscous coefficient of liquids of submicroliters [46]. According to Eq. (5.4), the optically induced driving torque can be readily calculated given parameters including power and wavelength of the laser and properties and size of the birefringent microparticle. As a result, the viscous drag force can be facilely measured from the balance of the driving optical torque and viscous torque.

How can we drive particles made of nonbirefringent materials? One approach is to rotate a birefringent microparticle to power the rotation of a nearby nonbirefrin-gent microgear structure as shown in Fig. 5.10A–B [43]. First, two microstructures made of silica and calcite, respectively, are held by two separate optical traps. When the mode of the trap laser is turned into circular polarization, the birefringent mic-roparticle starts to rotate, while the nonbirefringent silica microgear stays still. Next the optical traps are moved to bring the two microstructures next to each other. The silica microgear starts to rotate due to the surrounding flows generated by the spin-ning birefringent microstructure.

Another approach uses form birefringence where nonbirefringent materials are structured to exhibit birefringent characteristics to achieve the transfer of spin angular momentum to the micromachines. For example, as shown in Fig. 5.10C–D, in 2005 Neale and coworkers designed microgears made of nonbirefringent SU-8 polymer having photonic nanostructures that exhibit birefringent characteristics, which can be rotated by circularly polarized light [44]. The birefringent effect arises from the one-dimensional photonic crystal created on the microgear. When the polarization of light is parallel to the microribs, photons will primarily go along the high refractive-index

τ=±cε2wE021−coskdn0−ne,

1432 Optically enabled robotic micro/nanodevices

regions; when the polarization of light is perpendicular to the ribs, photons have to go through both materials, where the effective refraction index is the average of them. The distinct indices along the two directions induce the birefringence of the microgear. From Eq. (5.4), the torque is maximized when ( )( )− =kd n ncos 00 e . Thus,

the condition of obtaining efficient manipulation is π λ( )− = =d n nk2 40 e . Note that

the difference between n0 and ne is determined by the rib width and the gap distance between ribs. Therefore, by proper design, a laser beam can rotate the microgears at

coskdn0−ne=0

dn0−ne=π2k=λ4

FIGURE 5.10

(A,B) Schematic and optical images of a silica microgear driven by a birefringent particle that is rotated by circularly polarized light. (C,D) Schematic and SEM image of a structurally birefringent microgear driven by transfer of spin angular momentum during illumination.

Part A,B: Reproduced with permission Friese MEJ, Rubinsztein-Dunlop H, Gold J, Hagberg P, Hanstorp D.

Optically driven micromachine elements. Appl Phys Lett 2001;78(4):547–549 [43]. Copyright (2001) AIP

Publishing LLC. Part C,D: Reproduced with permission Neale SL, MacDonald MP, Dholakia K, Krauss TF.

All-optical control of microfluidic components using form birefringence. Nat Mater 2005;4(7):530–533 [44].

Copyright (2005) Nature Publishing Group.

144 CHAPTER 5 Electromagnetic wave

maximum efficiency. The demonstrated concept offers a general approach to drive micromachines made of various nonbirefringent materials via controlling the spin angular momentum of light.

As aforementioned, the orbital angular momentum (lħ) carried by a photon could be potentially greater than its spin angular momentum, which is ±ħ for circularly polarized light. Therefore, the control and transfer of OAM is promising for higher efficiency optical manipulation. When Allen and coworkers in 1994 found that a Laguerre-Gaussian (LG) laser mode has a well-defined OAM, they proposed ex-periments similar to that of Beth, to observe torques generated on suspended lenses caused by transfer of OAM [47]. Later He and coworkers made the first observation of the transfer of OAM by rotating a copper oxide microparticle by a linearly polar-ized LG mode laser, which does not carry spin angular momentum [48]. The transfer of angular momentum is due to the absorption of the microparticle, whose rota-tion direction can be determined by the helicity of the LG laser beam. As shown in Fig. 5.11A–B, laser beams carrying OAM have a helical wave front, which focus into a ring rather than a point [49,50]. These helical light modes, also known as optical vortices, exhibit ring intensity patterns that can facilely actuate micro/nanodevices [51]. As shown in Fig. 5.11C, micro/nanoobjects smaller than the ring are trapped in optical vortices and compelled to orbit around the ring pattern. The rotary torque (τ) on an absorbing particle is given by [52]:

τω

∝ Pl,a

(5.5)

where Pa is the absorbed laser power, w is the optical frequency, and l is topologi-cal charge. Owing to the linear dependence of the rotary torque (τ) on topological charge (l), dynamic holographic optical tweezers, which can create helical modes up to l = 200, is exceptionally powerful in generating optical vortices [53]. Fig. 5.11D shows a 3 × 3 array of microparticles trapped on the ring pattern in the optical vor-tices [54]. These trapped microparticles stir up circulating microflows as they rap-idly circle around the ring. By changing the intensities, distributions and topological charges of optical vortices in the array, the resulting flow field can be reconfigured dynamically. For instance, as shown in Fig. 5.11E, the microparticles are arranged in a 3 × 2 array, with opposite rotations in the upper and lower rows, forming into a microfluidic pump [55].

Besides the OAM transfer from a helical-mode laser to absorbing microparticles, a similar transfer occurs on a device that converts a plane wave into a helical wave. These light converters can be used to manipulate micro/nanodevices as shown in Fig. 5.12. Experimental demonstrations have shown an average OAM transfer as high as 34.55 ħ per photon. A turbine-like microrotor, of tens of microns in size, can be rotated with a high power conversion efficiency [58]. It can reach a speed of 500 rpm with a laser power less than 200 mW. Unlike fundamental studies of orbital angular momentum using linearly polarized helical mode lasers to avoid side effects from spin angular momentum, in practical applications, various micro/nanodevices are manipulated by combining both spin and orbital angular momenta [52,59,60].

τ∇Pawl,

1453 Plasmonically enabled robotic micro/nanodevices

3 PLASMONICALLY ENABLED ROBOTIC MICRO/NANODEVICESIn the last decade, plasmonic enabled nanomanipulation, which is based on coherent oscillation of charges in the conduction band of materials, have received intensive attention. The techniques can largely overcome drawbacks of conventional optical tweezers in the manipulation of micro/nanoobjects, including low spatial resolution

FIGURE 5.11 The Trapping and Rotation of Microparticles by Transfer of Orbital Angular Momentum

(A) Generation of helical light modes carrying orbital angular momentum. (B) Optical image of ring patterns of an optical vortex (central spot is an artifact). (C) Overlapped snapshots of a single microparticle trapped in an optical vortex and orbiting along the ring pattern. (D) Array of microstirrers using beads circulating in optical vortices. (E) Individually controllable circulating microspheres for microfluidic pumping (a single nanosphere highlighted in red moves 25 µm in 7 s to the left showing the pumping direction).

Part A: Reproduced with permission Grier DG. A revolution in optical manipulation. Nature

2003;424(6950):810–816 [49]. Copyright (2003) Nature Publishing Group; Part C: Reproduced with

permission Curtis JE, Grier DG. Structure of optical vortices. Phys Rev Lett 2003;90(13):133901 [56].

Copyright (2003) American Physical Society; Part D: Reproduced with permission Curtis JE, Koss BA, Grier

DG. Dynamic holographic optical tweezers. Optics Commun 2002;207(1–6):169–175 [57]. Copyright (2002)

Elsevier; Part E: Reproduced with permission Ladavac K, Grier DG. Microoptomechanical pumps assembled

and driven by holographic optical vortex arrays. Opt Express 2004;12(6):1144–1149 [55]. Copyright (2004)

Optical Society of America.

146 CHAPTER 5 Electromagnetic wave

and weak light-matter interactions, by significantly enhancing localized light inten-sity and breaking optical diffraction limit [62–64]. As shown in Fig. 5.13A,B, two types of surface plasmon have been applied in nanomanipulation: the surface plas-mon polariton (SPP) sustained at the surface of flat metallic film and localized sur-face plasmon (LSP) of metallic nanostructures with dimensions much smaller than the wavelength of incident light [65].

Since optical forces generated from surface plasmon polariton (SPP) on a homo-geneous metallic film is uniform, to obtain stable trapping of objects at a predefined

FIGURE 5.12 Rotation of a Turbine-Like Microrotor Driven by Orbital Angular Momentum When it Converts a Plane Wave Into Helical Wave

(A) Schematic of conversion of wave front. (B,C) Design and SEM image of a turbine-like microrotor. (D) Schematic diagram of interaction of light with the micromotor: A—refraction on the helicoid; B—internal reflection on the helicoid; C—refraction on the vertical wall.

Reproduced with permission Lin XF, Hu GQ, Chen QD, Niu LG, Li QS, Ostendorf A, et al. A light-driven

turbine-like micro-rotor and study on its light-to-mechanical power conversion efficiency. Appl Phys Lett

2012;101(11):113901 [61]. Copyright (2012) AIP Publishing LLC.

1473 Plasmonically enabled robotic micro/nanodevices

location requires patterning of the metal film to introduce a confined trapping well. As shown in Fig. 5.13C, Quidant et al. demonstrated plasmonic optical trapping of microbeads on Au microdisks by this method [62]. With arrays of microdisks much smaller than the spot size of the illumination laser, a large number of microbeads can be trapped simultaneously [66]. When the size of metallic nanostructures reduces to subwavelength region, localized surface plasmon dominates. Fig. 5.13D shows a design of plasmonic nanoantennas, where an ultrahigh field intensity is created at the nanogaps, allowing strong trapping of nanoobjects orders of magnitude smaller than those by the metallic microdisks [63].

In addition to trapping nanoentities with plasmonic effects, assisted with local-ized surface plasmon (LSP) resonance, dynamic manipulation of plasmonic nano-structures have also been exploited [67–72]. Similar to conventional optically driven

FIGURE 5.13 Plasmonic Optical Tweezers

Illustration of (A) surface plasmon polariton and (B) localized surface plasmon. (C) Schematic of trapping via plasmon polariton generated by Au microdisks. (D) Schematic of trapping via localized surface plasmon generated by Au nanoantennas.

Part A,B: Reproduced with permission Juan ML, Righini M, Quidant R. Plasmon nano-optical tweezers.

Nat Photon. 2011;5(6):349–356 [65]. Copyright (2011) Nature Publishing Group; Part C: Reproduced

with permission Righini M, Volpe G, Girard C, Petrov D, Quidant R. Surface plasmon optical tweezers:

tunable optical manipulation in the femtonewton range. Phys Rev Lett 2008;100(18):186804 [62].

Copyright (2008) American Physical Society. Part D: Reproduced with permission Grigorenko AN, Roberts

NW, Dickinson MR, ZhangY. Nanometric optical tweezers based on nanostructured substrates. Nat Photon

2008;2(6):365–370 [63]. Copyright (2008) Nature Publishing Group.

148 CHAPTER 5 Electromagnetic wave

micro/nanodevices, plasmonic nanostructures can be robotized by controlling either linear or angular photon momentum. A 2-µm-long T-shaped nanorotor made of as-sembled Au nanorods can be manipulated due to the anisotropic scattering of linear photon momenta as shown in Fig. 5.14A [67]. Although the overall length of the aggregate structure is a few micrometers, the individual Au nanorods are only 10 nm

FIGURE 5.14 Micro/Nano Rotary Motors Driven by Plasmonic Optical Tweezers

(A) Schematic and SEM image of a T-shaped plasmonic nanorotor made of assembled Au nanorods driven by redirection of linear optical momenta. (B) Schematic and TEM image of spinning Au nanoparticles driven by absorption of spin angular momenta. (C) Schematic and SEM images of Au nanorod motors driven by scattering of spin angular momenta. (D) Schematic and dark-field optical images of Ag nanowire rotors in an optical vortex driven by orbital angular momenta.

Part A: Reproduced with permission Jones PH, Palmisano F, Bonaccorso F, Gucciardi PG, Calogero G,

Ferrari AC, et al. Rotation Detection in Light-Driven Nanorotors. ACS Nano 2009;3(10):3077–3084 [67].

Copyright (2009) American Chemical Society; Part B: Reproduced with permission Lehmuskero A, Ogier

R, Gschneidtner T, Johansson P, Käll M. Ultrafast spinning of gold nanoparticles in water using circularly

polarized light. Nano Lett 2013;13(7):3129–3134 [68]. Copyright (2013) American Chemical Society.

Part C: Reproduced with permission Shao L, Yang Z-J, Andrén D, Johansson P, Käll M. Gold nanorod rotary

motors driven by resonant light scattering. ACS Nano 2015;9(12):12542–12551 [69]. Copyright (2015)

American Chemical Society; Part D: Reproduced with permission Yan Z, Scherer NF. Optical vortex induced

rotation of silver nanowires. J Phys Chem Lett 2013;4(17):2937–2942 [70]. Copyright (2013) American

Chemical Society.

1493 Plasmonically enabled robotic micro/nanodevices

in diameter and 45 nm in length. With laser excitation, strong plasmonic resonance can be generated in the Au nanorods, which results in substantially high forces and torques that rotate the T-shaped nanorotors. In contrast, individual symmetric Au nanorods just randomly move with laser excitation.

Plasmonic rotary nanorod motors driven by the transfer of spin angular mo-mentum via absorption or scattering are also reported as shown in Fig. 5.14B,C [68,69,72,73]. As shown in Fig. 5.14B, Au nanoparticles with a radius of ∼200 nm have been rotated at a frequency of several kilohertz due to the absorption of spin photon momentum. The rotation speed is much higher than any previously reported optical rotary motors [68]. The ultrahigh rotation speed of Au nanoparticles can be attributed to the significant enhanced local field intensity due to the plasmonic ef-fect and also the substantial laser induced heating efforts due to photon absorption, of which the former increases the rotation torques and the latter lowers viscosity of water. Although the laser induced heating efforts may reduce viscous drag force, it could generate undesired issues in various applications. Generation of plasmonic optical torques via plasmonic light scattering could potentially reduce the problem of laser induced heating. With this understanding, Au nanorods of reduced symmetry were rotated as nanomotors, of which the driving torques are due to light scattering resulted angular momentum transfer as shown in Fig. 5.14C [69]. The photon angular momentum transfer is maximized when the LSP resonances of Au nanorod coincides with the wavelength of the exciting laser. With only 10 mW, a nanorod can spin at a speed of ∼42 KHz. The ultrahigh speed and stable rotation with reduced heat genera-tion shows the great potential of the gold nanorod motors in various nanooptical-me-chanical devices. For demonstration purposes, the Au nanorod motors were applied in molecular sensing, where the presence of molecules in suspension can decrease the rotation speed of the microrotors not only by changing the liquid viscosity, but also by altering the liquid refractive index and thus shifting the plasmon resonance of the Au nanorods. Chapter 9 exploits laser-heated gold films to create flow in a novel light-driven syringe and Chapter 11 uses gold nanoparticles as light-controlled nanoheaters that can apply controlled perturbations on a cell membrane.

The orbital angular momentum of photons created in the optical vortex of Laguerre-Gauss beams was also demonstrated to rotate metallic nanowires motor as shown in Fig. 5.14D [70]. A linearly polarized laser beam, which does not carry spin angular momentum, was applied on a single silver nanowire with a diameter of ∼80 nm and a length over 10 µm. The rotation dynamics is governed by multiple forces, including the driving torques from the optical vortex, viscous drag from sus-pension, as well as plasmonic interactions of the nanorods with the laser beam. It is found that the rotation speed depends on the relative orientation of the nanorod and the polarization direction of the laser. The short dimension of the nanorod tends to plasmonically resonate and align with the polarization direction of the laser [73].

Based on the transfer of orbital angular momentum, a much smaller plasmonic nanomotor has been reported as shown in Fig. 5.15 [71]. With an asymmetric gam-madion gold nanostructure of 30 nm in thickness and 200 nm in diameter fabricat-ed by electric beam lithography as shown in Fig. 5.15A–B, nonuniform plasmonic

150 CHAPTER 5 Electromagnetic wave

modes at different components of the gammadion arise due to the interactions be-tween the incident light and the nanostructure. Note that the incident laser has no spin or orbital angular momentum. The phase retardation in the reemitted light due to the unique plasmonic modes generates a net orbital angular momentum to rotate the nanomotors. At a laser power of only 1 mW, the torques from the rotating nanomo-tors can be high enough to spin a silica microdisk. The rotation direction can be well controlled by the wavelength of the exciting laser.

4 ELECTRICALLY AND OPTOELECTRONICALLY ENABLED ROBOTIC MICRO/NANODEVICES4.1 ELECTRIC TWEEZERS ENABLED ROBOTIC MICRO/NANODEVICESThe electric tweezers technique is based on simultaneous application of DC and AC electric fields on nanoparticles [74–79]. The frequency of the AC electric field is in the range of kHz to MHz. Nanoentities suspended in a liquid medium experi-ence an electrophoretic (EP) force due to the DC field and dielectrophoretic (DEP) force due to the AC electric field. Electrophoretic force is due to Coulomb interac-tions of the charged outer electric double-layer (EDL) around the nanowires and the

FIGURE 5.15 Nanoscale Plasmonic Motors

(A,B) Schematic and SEM images of the plasmonic nanomotor. The motor is 100 nm in radius and 30 nm in thickness; the silica microdisk has an area of 2.2 × 2.2 µm and 600 nm in thickness. (C) Sequential dark-field images of a rotating silica microdisk driven by a plasmonic nanomotor.Reproduced with permission Liu M, Zentgraf T, Liu YM, Bartal G, Zhang X. Light-driven nanoscale plasmonic

motors. Nat Nanotechnol 2010;5(8):570–573 [71]. Copyright (2010) Nature Publishing Group.

1514 Electrically and optoelectronically enabled robotic micro/nanodevices

applied DC field. DEP force is due to the interaction between the nonuniform electric field (E) and the induced electric dipole moment (p) of the nanoparticle, given by FDEF = (p·∇)E [75,78]. Although the DEP force is zero in a uniform AC electric field, an electric torque, given by τ = ×p Ee , still can be applied to align nanowires in the direction of the AC field [75]. Therefore, the electric tweezers based on combined DC and AC electric fields can realize highly efficient and controllable manipulation: transporting nanoentities in the direction of a uniform DC field and aligning them in the direction of a uniform AC field as shown in Fig. 5.16A–B [74]. For instance, a nanowire functionalized with drugs can be transported along a prescribed path and deliver drugs onto a single live cell in the midst of many by the electric tweezers as shown in Fig. 5.16C–D [80]. Multiple nanowires can be transported to a single live cell sequentially to control the dose of a drug or to deliver multiple drugs.

As discussed earlier, a DEP force induced torque can align nanowires in the di-rection of an AC electric field, if we rotate the electric field, can we rotate nanowires [81,85]? As shown in Fig. 5.17A, a rotating electric field can be created via apply-ing four AC voltages with sequential 90° phase shift on a quadruple microelectrode [81]. When nanowires are randomly dispersed in the center of the quadruple mi-croelectrodes, they can freely rotate both clockwise and counterclockwise by sim-ply toggling the AC phase shift. The rotation speed (w) can be precisely controlled by the AC voltages (V), given by w ∼ V2. The V2 dependence is advantageous for achieving ultrahigh rotation speed. We have demonstrated rotation speeds up to 26000 rpm for a freely rotating Au nanowire (Fig. 5.17D) [72]. Nanowires made

τe=p×E

FIGURE 5.16 Electric Tweezers

(A,B) Schematics and overlaid sequential optical images of nanowires in translational motion using combined AC and DC electric fields with different configurations: (A) AC//DC; (B) AC⊥DC. (C,D) Schematic and overlaid sequential optical images of a robotized nanowire precisely transported to a designated live cell by the electric tweezers.Part A,B: Reproduced with permission Fan DL, Cammarata RC, Chien CL. Precision transport and assembling

of nanowires in suspension by electric fields. Appl Phys Lett 2008;92(9):093115 [74]. Copyright (2008) AIP

Publishing LLC; Part C,D: Reproduced with permission Fan DL, Zhu FQ, Cammarata RC, Chien CL. Electric

tweezers. Nano Today. 2011;6(4):339–354 [75]. Copyright (2011) Elsevier.

FIGURE 5.17 Rotary Nanomachines Operated by Electric Tweezers

(A) Schematic of rotation of freely suspended nanowire in the center of a quadruple microelectrode indicating the relative phases of the oscillating voltages. (B) Rotation of metallic (Au, Pt) and semiconducting (ZnO) nanowires, multiwall carbon nanotubes, and insulating (SiO2) nanotubes. (C) Schematic and overlaid sequential optical images of nanowire oscillators operated by the electric tweezers. (D) Schematic diagram of a rotary nanomotors with multiple integrated components operated by the electric tweezers. (E) Overlaid sequential optical images of an array of rotary nanomotors. (F,G) SEM images of a rotor and bearing used for assembling rotary nanomotors.

Part A,B: Reproduced with permission from Fan DL, Zhu FQ, Xu X, Cammarata RC, Chien CL.

Electronic properties of nanoentities revealed by electrically driven rotation. Proc Natl Acad Sci USA.

2012;109(24):9309–9313 [81]. Copyright (2012) National Academy of Sciences; Part C: Reproduced with

permission from Kim K, Zhu FQ, Fan D. Innovative mechanisms for precision assembly and actuation of

arrays of nanowire oscillators. ACS Nano 2013;7(4):3476–3483 [82]. Copyright (2013) American Chemical

Society; Part D: Reproduced with permission from Kim K, Guo J, Xu X, Fan D. Micromotors with step-motor

characteristics by controlled magnetic interactions among assembled components. ACS Nano 2015;9(1):548–

554 [83]. Copyright (2015) American Chemical Society; Part E–G: Reproduced with permission from Kim

K, Xu X, Guo J, Fan DL. Ultrahigh-speed rotating nanoelectromechanical system devices assembled from

nanoscale building blocks. Nat Commun 2014;5 [84]. Copyright (2014) Nature Publishing Group.

1534 Electrically and optoelectronically enabled robotic micro/nanodevices

of all types of materials, including metals, semiconductors, and insulators, can be rotated (Fig. 5.17B) [80].

To leverage the forces and torques produced by rotating nanowires for practi-cal applications, it is pivotal to anchor the positions of the nanowires during their rotation. Recently, an innovative type of rotary nanomachines assembled in arrays at designated locations was reported [82–84,86]. Unlike previously reported micro/nanomachines fabricated by top-down lithographic processes, the new rotary nano-machines are bottom-up assembled from nanoscale building blocks by using nanow-ires as rotors and nanomagnets as bearings as shown in Fig. 5.17C–D [82,84]. The nanowire rotors with embedded magnetic segment can be readily transported to the vicinity of nanobearings by the electric tweezers. The magnetic attractions between the nanowire and nanobearing precisely anchor the nanowire on the nanobearing, while still allow its rotation. Here, the magnetic attraction force is carefully adjusted by a designed spacer layer atop of the bearing. By using magnetic materials with unique perpendicular magnetic anisotropy, such as stressed Ni films, nanowire rotors can be aligned in the initial magnetic treatment direction and rotated to the direction of applied AC field. Therefore, as shown in Fig. 5.17C, a nanowire oscillator can be achieved by simply turning on and off the AC electric field alternatively. An array of nanowire oscillators can operate synchronously by toggling between two designed directions for more than 4000 cycles. The operation cycles number is comparable to cutting-edge devices made by complicated lithographical approaches. It demon-strates the great potential of using nanoparticles as active components for NEMS devices via precision and efficient nanomanipulation.

In another device demonstration, by applying a rotating AC electric field, the nanowire rotors can execute full round continuous rotation on the nanobearing, working as rotary nanomotors. The rotation is highly stable with controlled angle, speed, and orientation. With the advantageous highly efficient and controllable ma-nipulation by electric tweezers, these nanomotors can be facilely assembled into or-dered arrays, each taking ∼10 s. They rotate synchronously (Fig. 5.17E) to a speed at least 18,000 rpm at only 17 V. Furthermore, the nanomotors have all dimensions less than 1 µm, with a nanorotor of ∼900 nm in length, 200 nm in diameter, and nanobearings of ∼200 nm in diameter. Such nanomotors can also be made into nanoscale step-motors that rotate to desired angular positions by design [83]. Even more, the nanomotors exhibit extremely high robustness. With the use of Ti as the surface of the nanobearing, such nanomotors can rotate for 80 h over 1.1 million cycles [87].

This type of nanomotors finds prominent applications in nanosensing and drug releasing [88–90]. As shown in Fig. 5.18A–D, the nanomotors are designed and fab-ricated with surface distributed plasmonic-active Ag nanoparticles, which can readily detect biochemicals via surface enhanced Raman scattering (SERS). Specifically, the nanomotors consist of Au/Ni/Au nanowires as cores, silica as separation layers, and Ag nanoparticles uniformly distributed on the surfaces of the nanowires for SERS detection [88]. The plasmonic Ag nanoparticles have optimized sizes and junctions that provide a large number of hotspots (∼1200/µm2) and ultrasensitivity with an

154 CHAPTER 5 Electromagnetic wave

FIGURE 5.18 Plasmonic-Active Nanomotors Operated by the Electric Tweezers for SERS Sensing and Tuning Drug Release

(A–D) Schematics and TEM images of the plasmonic-active nanorotor structures. (E) Schematic of the assembled plasmonic-active nanomotor in rotation for tuning drug release and real-time detection. (F) Concentration of Nile Blue (NB) versus time at different rotation speeds. Inset: log-log plot of release rate versus rotation speed showing power dependence of 0.51.Part A–D: Reproduced with permission from Xu X, Kim K, Li H, Fan DL. Ordered arrays of raman nanosensors

for ultrasensitive and location predictable biochemical detection. Adv Mater 2012;24(40):5457–5463 [88].

Copyright (2012) John Wiley and Sons; Part E: Reproduced with permission from Kim K, Xu X, Guo J, Fan

DL. Ultrahigh-speed rotating nanoelectromechanical system devices assembled from nanoscale building

blocks. Nat Commun 2014;5 [84]. Copyright (2014) Nature Publishing Group; Part F: Reproduced with

permission from Xu X, Kim K, Fan D. Tunable release of multiplex biochemicals by plasmonically active rotary

nanomotors. Angew Chem Int Ed 2015;54(8):2525–2529 [90]. Copyright (2015) John Wiley and Sons.

1554 Electrically and optoelectronically enabled robotic micro/nanodevices

enhancement factor (EF) of 109–1010. Next, the plasmonic-active nanomotors are ma-nipulated, assembled into designed arrays, and rotated by the electric tweezers. The rotation can accurately tune the release rate of biochemicals as shown in Fig. 5.18E–F, which is monitored quantitatively in real-time by Raman spectroscopy [90]. It is found that the release rate of molecules (k) linearly increases with the square root of rotation speed (w), given by ωk ~ (inset Fig. 5.18F). Not only single molecules can be released; multiple molecules have been released from a single nanomotor fol-lowing the same dependence. This can be understood by the fluidic boundary layer theory, where the fluidic convection, proportional to rotation speed (w), monotoni-

cally reduces the thickness of the fluidic boundary layer (or diffusion layer, t) next

to the rotating nanowires, with a dependence of ω

t ~1

. For molecules diffusing

from the surface of nanomotors to bulk solution, the release rate (k) depends on the

thickness of the diffusion layer (t), given by t

~1. This matches the experimentally

obtained dependence of ωk ~ . Therefore, the experimental observation agrees with the fluidic boundary layer theory quantitatively. It is the first time to validate the fluidic boundary layer theory in nanoscale objects. Moreover, the newly discovered mechanism using mechanical rotation to control molecule release from the surface of nanoparticles is not limited to specific molecules. It can be applied to all types of biomolecules, such as drugs, cytokine, DNA, antigens, and antibodies. This work demonstrates a concept that changes the scheme of drug delivery and release from a passive to active fashion. It may remarkably impact the field of drug delivery, single-cell biology, and cell-cell communications.

4.2 OPTOELECTRONIC TWEEZERS ENABLED ROBOTIC MICRO/NANODEVICESOptoelectronic tweezers are a new optical manipulation technique that employs low-power light projections on photoconductive layers to create dynamically patterned microelectrodes that generate nonuniform electric fields and thus DEP forces to ma-nipulate micro/nanoobjects [91,92]. The minimum optical intensity required to gen-erate the “virtual electrodes” on the photoconductive layers is as low as 10 nW/µm2, which is 1/100,000 of those used in conventional optical tweezers. The schematic setup is shown in Fig. 5.19A, where an AC electric field is applied between two parallel electrodes: a transparent ITO glass and a surface photoconductive hydroge-nated amorphous silicon (a-Si:H) substrate [91,93]. When light is projected on the photoconductive a-Si:H substrate, the illuminated areas become conductive, forming into virtual microelectrodes. Therefore, electric fields can be strategically generated between the virtual microelectrodes patterned by light and the ITO glass. The electric fields can also be changed dynamically by varying the pattern of the projected light, which provide versatile micro/nanomanipulations. The manipulation resolution is determined by the size of the virtual electrodes. Unlike physical microelectrodes fabricated by various lithographical techniques, the ultimate resolution of virtual

k∼w

t∼1w

∼1t

k∼w

156 CHAPTER 5 Electromagnetic wave

electrodes is determined by projection precision of light and optical diffraction limit. Fig. 5.19B shows a demonstration of manipulation of nanoparticles by optoelec-tronic tweezers that can create 15,000 DEP traps at a time across an area of 1.3 mm2. The particles are trapped in the nonilluminated circular areas, where the electric field is weaker due to negative DEP forces. By dynamically varying the projected light patterns with digital micromirror devices (DMD), these trapped particles can be in-dividually manipulated simultaneously.

With the principle as discussed earlier, virtual micromachines can be created for continuous micro/nanomanipulation [91,94–96]. As shown in Fig. 5.20A, a micro-machine with integrated functions of optical conveyors, sorters, wedges, and joints can be achieved by the optoelectronic tweezers. Particles with different sizes are confined within the grid patterns, circled by the movement of the virtual machine, and separated by the asymmetric grids. Here that the particle’s path can be facilely changed by the dynamic wedge as shown in Fig. 5.20A. Furthermore, based on dis-tinct electric properties of micro/nanoobjects and their responses to electric fields, microparticles can be sorted according to their properties and functions. As shown

FIGURE 5.19 Optoelectronic Tweezers

(A) Schematic diagram of the optoelectronic tweezers. The electric field is generated between the virtual electrodes, created on the photoconductive Si electrode by DMD projected light patterns, and the counter ITO glass. Micro/nanoobjects are manipulated by the dynamic light patterns on the surface of the Si electrode. DMD: digital micromirror device; ITO: indium tin oxide. (B) Snapshots show largescale and parallel trapping and manipulation of individual microparticles.

Part A: Reproduced with permission from Wu MC. Optoelectronic tweezers. Nat Photon 2011;5(6):322–324

[92]. Copyright (2011) Nature Publishing Group; Part B: Reproduced with permission from Chiou PY, Ohta

AT, Wu MC. Massively parallel manipulation of single cells and microparticles using optical images. Nature

2005;436(7049):370–372 [91]. Copyright (2005) Nature Publishing Group.

1574 Electrically and optoelectronically enabled robotic micro/nanodevices

in Fig. 5.20B, live human B cells are concentrated and collected in the center of the smallest ring pattern from a mixture of live and dead cells.

Despite advantages in versatility and dynamic operation, the manipulation of microobjects with amorphous silicon based optoelectronic tweezers can only be performed in suspension with low electric conductivity due the large electrical re-sistance of the Si substrate even with photon illumination. Phototransistor-based optoelectronic tweezers were later developed to overcome this problem as shown in Fig. 5.21A–B [97]. The amorphous silicon layer in conventional optoelectronic tweezers is replaced by a pixelated phototransistor array. By adjusting the doping profile, the phototransistors are designed with ten times higher photon conductivity and 10 times lower dark-field conductivity than that of the cell culture medium. This design can enable successful switching on and off of the optoelectronic tweezers by light patterns even in highly conductive suspensions. With this setup, efficient trapping of live HeLa and Jurkat cells in Phosphate-Buffered Saline (PBS) and Dul-becco’s Modified Eagle’s Medium (DMEM) has been achieved. Another challenge for conventional optoelectronic tweezers is manipulating nanoscale objects, since these smaller particles exhibit stronger Brownian motion and receive weaker DEP forces. Zhang et al. reported lipid bilayer-integrated optoelectronic tweezers to ad-dress this challenge as shown in Fig. 5.21C [98]. An ultrathin (∼5 nm) 2D fluid lipid bilayer membrane is continuously covered on the photoconductive substrate. The lipid bilayer membrane confines the motion of nanoparticles by tethering them to the

FIGURE 5.20 Virtual Micromachines Enabled by the Optoelectronic Tweezers

(A) Schematic and optical image of virtual optical machines conveying and sorting particles by size. (B) Sequential optical images showing separation and collection of live cells from a mixture of live and dead cells when using a dynamic pattern.

Reproduced with permission from Chiou PY, Ohta AT, Wu MC. Massively parallel manipulation of single cells

and microparticles using optical images. Nature 2005;436(7049):370–372 [91]. Copyright (2005) Nature

Publishing Group.

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membrane in the vicinity of the substrate, where the highest electric field gradient exists. With the confinement efforts, dynamic and reversible in-plane manipulation of hundreds of gold nanoparticles of 60 nm diameter can be accomplished. Both im-provements can broaden the applications of the optoelectronic tweezers.

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FIGURE 5.21 Further Development of the Optoelectronic Tweezers

(A) Schematic of the phototransistor-based optoelectronic tweezers. (B) Optical image of array of cells captured by the phototransistor-based optoelectronic tweezers. (C) Schematic of the optoelectronic tweezers integrated with a supported lipid bilayer.

Part A,B: Reproduced with permission from Wu MC. Optoelectronic tweezers. Nat Photon 2011;5(6):322–

324 [92]. Copyright (2011) Nature Publishing Group; Part C: Reproduced with permission Ota S, Wang S,

Wang Y, Yin X, Zhang X. Lipid bilayer-integrated optoelectronic tweezers for nanoparticle manipulations. Nano

Lett 2013;13(6):2766–2770 [98]. Copyright (2013) American Chemical Society.

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CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00006-9Copyright © 2017 Elsevier Ltd. All rights reserved.

Gaszton Vizsnyiczai, Badri L. Aekbote, András Buzás, Pál Ormos, Lóránd KelemenBiological Research Centre, Szeged, Hungary

CHAPTER OUTLINE

1 Introduction ...................................................................................................... 1671.1 Two-Photon Polymerization .............................................................1691.2 Optical Manipulation ......................................................................170

2 Applications ..................................................................................................... 1722.1 Nonfunctionalized Structures ..........................................................1722.2 Functionalized Structures ...............................................................182

3 Conclusion and Outlook ..................................................................................... 186References .............................................................................................................. 186

1 INTRODUCTIONMechanical manipulation, actuation, or sensing on the micrometer and nanometer scale is becoming an important tool in bionanotechnology. With the advent of micro-fluidics, the examination area of exciting biological processes shrank down to this scale. In these tasks the target objects need to be moved from one location to another, its orientation needs to be altered, its shape needs to be changed and the correspond-ing responses need to be monitored from the microscopic to the nanoscopic level, ei-ther mechanical or biochemical. Localized detection of an analyte is also among the tasks that have to be carried out on the micrometer scale. For such manipulation or detection tasks, a multitude of tools are available from micropipettes, through mag-netic particles to AFM [1–3] or using methods that MEMS technologies can provide [4,5]. The applied forces that are meaningful in micrometer-sized systems are below the nanoNewton regime. However, it is often desired that the targeted objects have no hard-mechanical contact with the actuator. In this case external fields, such as acoustic [6], electric [7], magnetic [8], or optical are used to actuate the target objects directly, or through some intermediate tool. In these manipulation schemes the force range can reach down to the pN-fN regime.

Complex polymer microtools for on-demand contact-free applications

6

168 CHAPTER 6 Complex polymer microtools

Optical micromanipulation, the topic of this work has been successfully used to study the function or characteristics of biological macromolecules, proteins, DNA [9–13], cell organelles [14–16], or entire cells [17–21]. In these experiments the objects to be tested were manipulated either directly, or indirectly; in this latter case optical field was acting on a spherical dielectric bead and this was mechani-cally coupled to the objects to be manipulated. Spherical beads are practical due to their ease of use and well-established characterization procedures. The application of microtools with complex shape would allow much more elaborate manipulation schemes, new types of movements would be possible. Microbeads are classically used in manipulation experiments for translation, but when these objects are made of birefringent material (for instance, vaterite [22]) or to have a quasispherical shape (shape birefringence [23]), they can be rotated with a speed even up to 5 MHz [24]. Complex tools offer a further option to generate rotational motion to realize its total positional control thereby extending the possibilities for indirect manipulation. Also, due to their extended size, the point of action and the point where it interacts with the optical field can be separated to an arbitrary distance so that a possibly damaging interaction between the object and strong laser light can be prevented.

Microfluidics systems, a rapidly developing technology for integrating and miniatur-izing complete chemical-biological procedures, are also the subject to micron-scale op-tical manipulation. While the basic liquid-handling tasks are carried out with established methods (valves, pumps, filters) [25] often local manipulation/perturbation is needed, like local pumping of minute (pL) volumes or stirring, etc. [26–28]. Optically manipu-lated complex microtools offer such applications too, as shown later in this chapter.

There are numerous ways to prepare extended microstructures of special shape; the particular optimal procedure depends on the required material and spatial resolution. Optical or electron beam lithography can easily produce 2D objects [29]. In order to make 3D objects mask lithography techniques require rigorous, multistep mask align-ment procedures. However, with laser direct writing techniques, the preparation occurs usually in single step, where a focused laser beam is translated across the production volume [30]. The resolution, or feature size of the microstructures produced by for in-stance electron beam lithography can be as good as a few nanometers [31], while with optical methods it is down to ∼50 nm [32]. It shows the popularity of the technique that such systems are already commercially available (Nanoscribe GmbH, Germany).

Another very important aspect of the polymer microtools is that they can be func-tionalized with specific physical, chemical, and biochemical treatments. These pro-cesses largely extend the capabilities of the tools and enable their targeted specific interaction with various types of objects. The surfaces can be coated with metal in the forms of plane film or nanoparticles, with simple molecules, larger biological macromolecules, or even cells can be attached to them.

In this chapter, we will survey results on microstructures produced by direct la-ser writing applying two-photon polymerization with special attention to their light-based manipulation and possible applications. In the wide range of the demonstrated applications all the introduced polymerized complex tools are actuated either by ra-diation pressure, or optical trapping, or with the combination of the two.

1691 Introduction

1.1 TWO-PHOTON POLYMERIZATIONTwo-photon polymerization (TPP) is an established method to prepare 3D structures with feature size in the range of a few 100 nm [33]. With special optical arrangement (STED), however, this figure can be reduced to about 50 nm [34,35]. TPP is based on the illumination of a photocurable material with a beam of an ultrafast laser source tightly focused by a high NA microscope objective (Fig. 6.1). When the material and the focal spot are moved relative to each other, a well-defined trajectory is il-luminated where polymerization can take place. Typical laser powers are in the few milliWatt region and the focal spot translation speed can reach up to millimeter per second. The material undergoes a liquid to solid phase transition as a consequence of the irradiation (and, in some cases of a subsequent heat-treatment), and the nonpo-lymerized liquid can easily be removed from the sample. Eventually, solidified, real 3D structures remain on the substrate as free-standing microobjects. The size of the polymerized features depends mainly on the optical characteristics of the system. The facts that the probability of two-photon absorption depends quadratically on the intensity, and that TPP is a threshold effect ensures that features smaller than the dif-fraction limit can be achieved around the focal spot.

The procedure of laser writing is based on the relative motion of the polymer-ized material and the focal spot along a predetermined trajectory. There are different realizations of this procedure; the most obvious cases are when the light is scanned mechanically, or the material is scanned with an xyz piezo positioner [33]. The ap-plication of diffractive optical elements [36] can improve the efficiency of the proce-dure. Recently, Vizsnyiczai and coworkers [37] introduced a very efficient procedure where TPP scanning is achieved without any mechanical motion, using patterned light shaped by a spatial light modulator.

The commercialization of TPP (Nanoscribe’s system) resulted that its applica-tions now extends from photonics through microengineering to biology. The struc-tures can be fixed to the substrate surface where they were created or removed from

FIGURE 6.1 Schematics of the 3D Direct Writing by Two-Photon Polymerization

170 CHAPTER 6 Complex polymer microtools

it and used elsewhere. Classical application of TPP is the construction of photonic crystal structures with precisely tailored optical characteristics, such as reflection or transmission [38]. These characteristics are determined by the material refractive index and most importantly by the geometrical characteristics of the 3D lattices. For example, reflection spectra specifically designed to have maxima between 450 and 600 nm was made by polymerizing woodpile structures of 20 × 20 µm lateral dimen-sions with rod spacing of 300–400 nm [34]. Surface-attached structures are also de-veloped for sensing applications, for instance in the form of microdisc resonators and integrated waveguides [39,40]. Recent results demonstrate the manufacture of high aspect ratio structures with TPP using low NA objectives [41]. In this case the lateral resolution is sacrificed to reach structure height of several millimeters. It is also pos-sible to create combined structures with fixed and movable parts. The application of such tools is usually joined with a method that enables the controlled actuation of the moving part. Due to the size of the structures and the frequent requirement of nonin-vasiveness, optical actuation methods are popular. There are examples for radiation pressure-driven tools [42,43] as well as ones actuated by laser tweezers [44,45], or the combination of the two [26,28,46,47].

1.2 OPTICAL MANIPULATION1.2.1 Actuating the structuresThe basis of optical manipulation is that the momentum of light changes during the light-matter interaction due to absorption or scattering. The relevant phenomena are well understood and discussed in detail, for example, in [48–51] and others; the basics of optical trapping systems are reviewed in Chapter 1. The object to be ma-nipulated needs to be either absorptive at the wavelength of the manipulating light or its refractive index different from that of the surrounding medium. The change of the direction and magnitude of the wave changes the flux of momentum associated with its photons. The interaction force can manifest in various ways propelling the structures forward, rotating them or, keeping them locked at a given position. We note here that absorption is rarely used for optical manipulation, because the ab-sorbed photon energy is converted mostly to heat and the resulting heating makes the actuation extremely hard to control. Reflection always occurs on the medium-object interface to an extent determined by their relative refractive indices at the light’s wavelength in the medium and the angle of incidence given by the Fresnel formulae. Here, the momentum change exerts a force toward the other medium, normal to the reflective surface, its amplitude depends on the surface’s reflectance. Except for per-fectly reflecting surfaces, refraction is also always present resulting in changing the direction of propagation, which also exerts a force normal to the interface and toward the lower-index material.

The simplest way to optically manipulate microstructures is using radiation pres-sure which may originate from absorption, scattering, diffraction, or reflection of the light. Here the momentum change is converted to translation or rotation, no posi-tion clamping takes place. In spite of the triviality of the effect, many applications

1711 Introduction

with cleverly designed microtools can effectively and controllably take advantage of it [43,52]. Special cases for helical structures are discussed by Gauthier et al [42,53]. Fixing the position of the microstructures can be achieved by optical trap-ping (OT) that requires more complex optical systems. Furthermore, OT can control the structures’ actuation more precisely giving rise to more manipulation schemes: 6 degrees of freedom actuation is possible with exactly defined speed. Naturally, ra-diation pressure and optical trapping can be combined into remarkable experiments that demonstrates micromechanical systems assembly [26,47,54], stirring [23,28], or particle sorting [55] just to name a few.

Optical trapping that provides reliable positioning accuracy became possible only after the invention of the laser. The pioneer experiments were performed by Ashkin: in his first experiment beads were translated linearly, trapped in counter-propagating two-beam traps [56] already permitting 3D positioning. Later the single-beam ar-rangement was also introduced [57]. Here a sufficiently high NA microscope objec-tive focuses the trapping light. The two main forces that govern optical trapping are the gradient force that drives the structures toward higher light intensity and the scat-tering force that tries to repel them in the direction of the light propagation. Stable trapping in a single beam optical trap can only be performed if the gradient force exceeds the scattering one.

In the majority of cases, the single objective arrangement is used to create one or more trapping focal spots where the structures are held or moved around. Be-sides this, however, arrangements using two counter-propagating, weakly focused laser beams were also developed and used for stable 3D positioning of extended microstructures. Here, the main advantage is that high NA objectives are not needed and large working distance objectives permit large range of manipulation distance to cover larger distances for the manipulation [58] with the cost of small trapping force along the optical axis.

For practical purposes it is very important that using a single focusing objective does not mean that one is restricted to only one focused beam, therefore one trap-ping focal spot. The presented microtools are complex so multiple trapping beams are needed for their manipulation. For this, time-shared OT [59] or holographic OT (HOT) are the most frequently used ones [45,60,61]. In fact, in the case of the time-shared method there is only one beam at every moment, but its location is switched fast between the predefined focal positions, so that the trap returns to the spot before the trapped bead could move away significantly due to diffusion. On the other hand, holographic optical tweezers are using a diffractive optical element (DOE) to realize several focal spots simultaneously that are present in the sample chamber at the same time. The most popular DOE is a dynamically reconfigurable phase-manipulation element, the spatial light modulator (SLM) that manipulate the phase-front of the beam in an arbitrary way. It can split the incoming bam into several beams, deviate them, change their divergence and even can add orbital angular momentum to the beam, just to name a few, practically interesting possibilities. HOT allows for the parallel 3D manipulation of multiple beads or that of a single complex 3D object as illustrated in Fig. 6.2.

172 CHAPTER 6 Complex polymer microtools

Overall, the design of the microstructure is determined by the targeted application and the possibilities of driving the microtool. It is the actuation of the freely mov-able, complex 3D microtools that require one of the most advanced manipulation scheme with multiple trapping foci moved around in the trapping region. In case of these tools the interaction point with the laser foci are limited to certain parts, to the “handles,” while the rest of the structure is not illuminated. This allows the un-disturbed interaction of these nonilluminated parts with light-sensitive or absorbing target objects. The design considerations of such structures are discussed by [62–64].

2 APPLICATIONS2.1 NONFUNCTIONALIZED STRUCTURES2.1.1 Radiation pressure-driven actuationFirst we discuss applications that use only radiation pressure to actuate microtools in a fluid environment. Its relative simplicity and, in many cases, the higher degree of parallelization makes the method attractive when particles are to be translated only by light in a closed environment.

Self-propelled microscopic robots [65] are developed toward technological ap-plications primarily in the biomedical field, for tasks, such as material transport. The collective motion of active biological systems, for example, bacteria colony or a flock of larger animals, is also the target of a wide range of studies due to the systems’ remarkable dynamical phenomena. The study of birds flying in flocks, for instance, has relevance in the understanding of the interactions among group members and the characteristic parameters that describe these interactions (number of members involved or delay time of the resulted action) [66]. These parameters may even sug-gest such evolutionary perspectives that for smaller travelling groups hierarchical

FIGURE 6.2 Optical Microscopic Image of Three Trapping Focal Spots Created by a Holographic Optical Tweezers Setup and a Two-Photon Polymerized Microstructure (its SEM Image in the Insert) That is Manipulated by the Focal Spots

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organization is favored over the egalitarian one. In the micro world, the collective mo-tion within bacterial colonies [67,68] to find food, light, or just freely available space may determine their capability of conquering new habitats that has great relevance for medicine. Bacterial motion can even be exploited to create biohybrid microrobots that attach them to artificial objects for actuation, as discussed in Chapter 14. While the moving organisms themselves represent the essential features, if specific theories are to be tested there is a need for model systems with controlled and variable prop-erties so that detailed physical description is possible [69–72]. Buzás and coworkers introduced a very effective self-propelled swimmer that simply uses unfocused light for motion [69]. They move in a two-dimensional system on a planar surface. The two requirements concerning propulsion are that it should move the units in a regular fashion and in a direction determined only by the body of the swimmer. This require-ment is very different from regular optical manipulation, where the motion occurs either along the direction of light—when light pushes the objects, or the object is moved by moving the position of the trap itself. The authors used reflective wedge-shaped particles, sliding on a flat horizontal surface (that was actually the bottom of a hanging drop of water in air) (Fig. 6.3A–C). The 10 µm long particles were made by TPP and coated by a thin layer of gold. The area where the wedges could move was illuminated from above in a direction perpendicular to the surface of motion by a collimated homogeneous light beam. The structure was designed such that it always rests on one plane of the wedge so the light coming from above is reflected to the side from the other plane transferring its momentum to the body. The momentum change during reflection provides a force sufficient to propel the particles, resulting in a motion in a direction determined only by their shape and orientation. The observed correlation of the direction of motion with the orientation of the swimmers was high: the majority of the particles moved in the direction within ±30 degree relative to their long axis with the persistence length of 355 µm.

Another self-propelled, autonomously moving particle was demonstrated by Oroszi and coworkers [73]. In this case the object was a rotor, also moving on a horizontal surface in a direction determined by its position when illuminated from above. This work, in addition to provide a new quasiautonomous robot system, was also aimed to study theoretically and experimentally the conditions of rotation of rotor-shaped objects induced by collimated light carrying no angular momentum, when the axis of rotation is perpendicular to the direction of illumination. A system-atic modeling was carried out studying rotors of homogeneous, primarily nonabsorb-ing material, either transparent or reflective. The illumination was a collimated light beam of homogeneous intensity distribution, fully covering the objects. The rotors were of two basic types: one is prism-like shapes, that is, 3D objects that are created by the extrusion of a 2D footprint, and the other is true 3D objects having a varying structure along the rotation axis.

In the case of prism-like rotors, the problem is two-dimensional: light scatter-ing occurs only in the plane perpendicular to the rotation axis. Modeling a large number of different shapes it was found that in the lossless case (no absorption), sustained rotation is not possible neither for refractive nor for reflective objects.

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Although in certain orientations light exerts torque on the object, it eventually ends up in an angular trap. On the other hand, if the rotors partially absorb light, rotation becomes possible for certain rotor shapes and absorption ranges. It was further found, that if the structure has true 3D shape so that scattering also hap-pens in three dimensions, rotation becomes possible. The key feature here is that during 3D scattering light is extracted from the two-dimensional plane. To dem-onstrate this effect, a true three-dimensional object was designed where the scat-tering process is three-dimensional. It has fourfold symmetry, and it is based on a cross-based prism structure. The structure is deformed with a twist of angle Θ symmetrically in opposite directions along the two halves of the rotation axis (Figure 6.3D) and fits into a cylinder of equal height and diameter. With these

FIGURE 6.3

Scanning electron (A) and (B) and optical microscopic images of the swimmers used by Buzás and coworkers [69]. The images in (C) are from the actual experiments. The SEM image of the rolling 3D rotor used by Oroszi and coworkers (D) [73], illustrating the torsion angle Θ, which is the angle between the planes defined by the solid and dashed lines.

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constraints, a single torsion angle parameter fully determines the geometry and consequently the torque exerted by light reflected form the surfaces. According to the simulations, this type of rotor can exhibit sustained rotation in a wide range of torsion parameter values.

Experimentally, the rotation of such a design was demonstrated. A design was selected with a torsion of 105 degrees. The structure was made by TPP and coated with gold to ensure high reflectivity. The structures indeed, exhibited stable rotation that manifested in rolling on a flat surface into the direction perpendicular to their axis of rotation. These experiments supported that true 3D objects can be rotated by a collimated beam of light propagating perpendicular to their axis of rotation. These studies also resulted in a general physical statement: light cannot induce rotation if such a perpendicularly illuminated system is lossless and the scattering process is two-dimensional. Further discussions on rotating micromachines can be found in Chapter 4.

Optical manipulation techniques may also find application in microfluidic de-vices, such as actuators, pumps, and valves. Kelemen and coworkers describe a fully integrated optically driven micromotor, illustrated on Fig. 6.4, that can work inde-pendently even without a microscope [43]. This motor represents a fundamental ele-ment of optically controlled microfluidic devices that can act as a pump or can even drive other movable parts. The entire system, built on the surface of a microscope cover slip, consisted of a cogwheel-shaped rotor held by a stable-axis structure and of an optical waveguide attached onto the glass. The mutual position of the motor and the waveguide was such that when light emerged from the waveguide it hit the rotor in the tangential direction. The motor with the moving and static parts and the waveguide were both built by laser-assisted polymerization. The motor diameter was 10 µm, so was the width of the waveguide; its length was, however, several millime-ters. A 532 nm wavelength laser was coupled into the far end of the waveguide and the motor was running as expected.

FIGURE 6.4

Schematic drawing (A) and electronmicrograph (B) of the integrated micromotor polymerized on top of a microscope cover slip [43].

176 CHAPTER 6 Complex polymer microtools

The action of the rotor is primarily due to the reflection of the light from its sur-faces considering the refractive index difference of the surrounding water (n = 1.33) and that of the used material (SU8 polymer, n = 1.6). Assuming planar rotor blade surfaces and taking into account the reflections on five blades of different angles with respect to the direction of the driving light, the resulting torque was estimated to be about 6 × 10−18 Nm at the maximum intensity (25 mW laser power). At this maximum illumination, the rotation speed was about 2 Hz. The torque due to the viscous drag was also calculated for such a spinning rotor, and was found to be about 19 × 10−18 Nm ± 20%. This value is in the range typical for systems of micrometer dimension and considering the used approximations reasonably matches the one calculated for the driving. Also comparing this value with those measured on other propeller sys-tems driven in optical tweezers [47] or on flat particles oriented by tweezers formed by linearly polarized light [74], we find them comparable. The main advantage of this motor is that it is a stand-alone system. Once light is coupled into the waveguide, it is completely independent of any microscope and, therefore it can be used as driving units of lab-on-a-chip systems with optical control and optical actuation.

The theoretical analysis of such a light-driven micromotor was carried out by Metzger and coworkers [75] to evaluate its expected performance. The model pre-dicts the torques on the motor and enables the authors to carry out a theoretical analysis of the light source-motor system and optimize the micromotor geometry. The theoretical model comprises of a monochromatic laser field of wavelength λ propagating toward the motor (z axis) originating from a single-mode fiber. The beam waist is located at the end facet of the fiber and separated by a distance z from the outer rim of the micromotor. The center of the beam is offset by x from the axis of rotation of the motor. The field is modeled as a Gaussian beam of spotsize w0 emerg-ing from the fiber facet and power P. It interacts with a micromotor of refractive index nc = 1.58 (material SU8, approximated to a wavelength of 1070 nm) immersed in a host medium (water) of refractive-index nh = 1.33.

The benefit of the model is that micromotor systems can be simply evaluated and their performance improved and new systems can be designed by systematically testing several key geometric and photonic parameters. Modeling four different rotor geom-etries, the theory nonsurprisingly predicted that the torque drops with the distance of the fiber’s facet and the rotor, but even at the distance of about 100 µm it drops to only about half of the maximum observed at zero distance when the offset was 5.5 µm. For two of the four tested motor designs, where there were distinct fans for the rotors, the torque showed distinct maxima with the offset of around 3–4 µm. For application point of view, it was important to learn that slight geometrical changes of the design can have an almost twofold increase in the torque, and that the system is sensitive with microm-eter precision to the relative positioning of the driving beam and the rotor center.

2.1.2 Actuation of fixed structures with OTAs mentioned before, optical trapping allows for more precise actuation of micro-scopic structures than radiation pressure, since their position can be set with sub-micrometer accuracy. This is often exploited for surface-attached structures where

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the functional parts are moving around an axis, similarly as in the previous section. Maruo and coworkers used this scheme to move mechanical microtweezers on a pair of axes for gripping and microturbine systems for mechanical driving [76,77]. The microtweezers with submicrometer tips were planned to be used to grasp microscale objects such as micro beads or cells in liquid environment. One advantage in contrast to the direct optical trapping of a target sample, is that the focus of the trapping laser beam does not irradiate the target object, eliminating the risk of irradiation damage. The torque exerted by the ∼7 µm long manipulator arms were controlled simply by positioning the trapping beam of constant power along the arm and was estimated to be between 0.4 and 1.5 × 10−19 Nm. The other structure was a microturbine of 7 µm radius that rotated around an axis, and its blades were trapped by optical tweezers for actuation (laser power: 200 mW). The turbine’s rotation was carried out by moving the optical tweezers around in a circle, reaching a speed of 4.6 RPM with an estimat-ed 3.3 × 10−17 Nm torque. The combination of several types of the described micro-mechanical parts envisions the creation of advanced optically driven microdevices for tasks, such as cell stimulation or microanalysis systems for single molecules.

An elegant, more recent work demonstrates how such an axis-constrained mi-crotool can serve not as a micromanipulator but as a sensor in a microfluidic envi-ronment. Liu and coworkers [78] polymerized a micrometer-sized flow meter that rotates according to the velocity of the liquid flow around it. The body of the meter is connected to the axis through a very thin polymer rod that serves as a torsion spring generating the restoring force, and the rotation angle of the body indicates the flow velocity. Here the authors use a single beam optical tweezers to calibrate the rotation angle to the force applied by the rod spring.

2.1.3 Actuation of free-floating structures with a single-beam OTProbably in the vast majority of the applications the optically manipulated objects are to be detached from the sample substrate or from any kind of axis or supporting structure and moved freely around in the working environment. Historically, the first such microtools were single beads that provided a wealth of information on biologi-cal macromolecules, such as DNA [79,80] and proteins [10,13,81] or even viruses [82] and cells [83,84]. Unfortunately, these simple beads cannot be rotated, unless special material and trapping beam is used (birefringent particle (vaterite) trapped by circularly polarized light [22,24]). Extended structures trapped by a single beam can overcome this difficulty and display additional functionalities. The first artifi-cial microstructure, that rotates relatively fast while trapped was shown by Higu-rashi and coworkers [54] using SiO2 cross-shaped particles. Better quality and more elaborately-shaped objects were introduced by Galajda and Ormos [47,85] who used laser-induced 3D direct writing to make helical rotors to trap and generate torque for further use or by Asavei and coworkers [23] who produced shape-birefringent quasi-spheres for laser-induced rotation.

The Ormos group tried different shapes and found that the rotation efficiency and a stable position in the trap are both important requirements for a useful shape [85]. In order to realize stable trapping a central linear axis was necessary to be added to

178 CHAPTER 6 Complex polymer microtools

the rotor. The rotation of these rotors was more efficient than reported earlier: with 20 mW laser power several Hz speed was achieved. Interestingly, the angular rotation was not dependent on the polarization state of the trapping light, but was linearly pro-portional to the light intensity. Due to the shape of the structure, it could be trapped in two stable positions: the axis of the rotor was aligned along the optical axis in both cases, but it pointed toward or opposite to the propagation of the light. The efficiency of the rotation was largely dependent on the rotor’s orientation. The achieved torque was in the range of 3 × 10−17 Nm. Quite importantly, the authors also demonstrated that such a free-floating rotor when trapped and rotated can induce rotation on cog-wheels fixed onto an axis that is attached to the substrate. This example nicely shows that driving rotors and mechanisms of practically arbitrary complexity can be put together in the same compartment.

Galajda and Ormos [86] also designed and built light driven rotors that could be trapped and driven in high NA optical traps the rotation direction of which could be reversed. The rotors were driven by the component of the light that is perpendicular to the propagation direction. This light component changes sign before and behind the focus: before the focus it points inwards, toward the optical axis while after the focus it point away from the axis. Consequently, the rotor rotation changes direction when positioned before or behind the focus.

The Rubinsztein-Dunlop group [23] polymerized shape-birefringent particles in the form of microsphere with cylindrical cavities (holey spheres). While a simple polymer sphere does not rotate when trapped, the cavities break this symmetry and rotation will be possible. Using circularly polarized light carrying spin angular mo-mentum as trapping beam rotation of these spheres was successfully demonstrated with up to 2 Hz. The analysis of the observed motion revealed that spheres with only one hole rotate with an eccentric motion, while for an improved sphere design with two holes this eccentric behavior vanished and steady rotation around the center of their mass was achieved.

Microfluidics can also benefit from optically trapped freely-floating microstruc-tures. Maruo and coworkers introduced a micro pump that can propel fluid in mi-crofluidic environment with 18 pL/min speed [26]. The micropump consisted of a spinning microrotor with twin spiral blades of the type introduced by Galajda and Ormos [86] and is confined to a U-shaped microchannel where it generates a lami-nar flow. When the twin spiral microrotor was optically trapped it stably rotated at a speed of up to 500 RPM and it maintained its 3D position. Using twin blades the authors improved the torque that can be generated with the tool. It can be understood if we consider that the optical tweezers are holding the structure roughly at its center, and both the converging and the diverging part of the focused trapping beam scatters from the blades of opposite helicity giving the torque of the same sense.

Two-photon polymerized, extended, trapped structures may be used not only to drive mechanical systems or pump liquid, but can also represent model systems for biological phenomena. Hydrodynamic synchronization is a fundamental physi-cal phenomenon that is suggested to play a role also in biological systems, such as the emergence of synchronous behavior in the motion of microscopic organs, for

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example, bacterial flagella or cilia. During the phenomena self-sustained oscillators communicate through perturbations in the surrounding fluid and converge to a stable synchronized state. DiLeonardo and colleagues [87] used a pair of light driven ro-tors to study the synchronization in a microscopic model system. Helical, threefold symmetry TPP micropropellers of opposite circularity were produced and driven by radiation pressure (Fig. 6.5A). When a pair of such rotors was trapped, they were holding a stable position and spinning in opposite directions. By changing the loca-tion and power of each trap, the rotors’ position and applied optical torques could be controlled independently. To observe synchronization the = θ1 − θ2 relative angle of the two rotors (Fig. 6.5B) was monitored during rotation. When the rotor centers were separated by 6 µm, and the relative torques changed gradually (one increased, the other reduced), there was a tendency for the relative phase to get locked at values of ∼60 degree modulo 120 degree meaning that the arms of the rotors stayed as close to each other as possible (Fig. 6.5C). This synchronization effect was found to be very sensitive to the rotors distance and to the relative torque: for 7 µm distance the phase locking was much weaker. It could also be visualized by plotting the probabil-ity distribution of at various relative torque values; this function showed a distinct peak at 60 degree for small torque differences and flattened out for larger ones.

FIGURE 6.5

Scanning electron micrograph of the rotors used to study hydrodynamic synchronization (A) [87]. The relative angle was calculated from the angles of the two rotors: = θ1−θ2 as shown on the optical microscope image on (B). The relative angle as the function of time for 6 µm rotor separation (C); the phase lock at 60 degree modulo 120 degree was more pronounced for small M1−M2 relative torques.

180 CHAPTER 6 Complex polymer microtools

The synchronization was modeled numerically by regarding each rotor as a rigid assembly of spherical beads. Each bead was subject to a force resulting from the in-ternal mechanical interaction with neighboring beads plus the external optical force. Hydrodynamic interactions were accounted for by assuming that each bead is ad-vected by a background flow that is obtained as the sum of the Stokes flows propagat-ing from point forces acting on all the other beads. The authors wrote the equation of motion for the one cycle average of the phase difference ϕ in the form of

ϕ ϕϕ

= − + ∆

RdU

dM

( )

where ∆M = |M1|−|M2| is the applied torque difference, R is the rotational mobility of an isolated rotor, and U is a periodic potential for encoding the whole complex structure of hydrodynamic interactions. Calculations show that a maximum in the probability distribution corresponds to a minimum at about 60 degree in the poten-tial U. it was also found that the rotors move as overdamped particles over a tilted periodic potential U−R·∆M·. For the highest torque mismatches, the potentials are so much tilted that there’s no longer a stable equilibrium position, although a peak is still visible in the corresponding P(). This is due to the fact that, even if the phase lag is not getting locked in local minima, it spends more time in the neighborhood of 60 degree, where the slope of the tilted potential is minimum. It was also an im-portant finding that there is a link between synchronized states and extrema in the energy dissipation function. Whether synchronization corresponds to minimum or maximum dissipation can depend critically on the detailed shape of the rotors.

2.1.4 Actuation with multibeam OTIn this part we introduce applications for extended 3D structures whose position was stabilized or moved freely in 3D using multiple trapping beams. Neither task is simple, as investigated by many using readily available probe objects (such as a dia-tom in [88]) or polymer microstructures [45,89]. While the full control requires the precise knowledge of the three-dimensional position of the sample, in many applica-tions the lateral 2D information is sufficient. However, Phillips and coworkers stud-ied the problem in 3D with specific microstructures using stereo-observation [63]. The analysis of the holographically trapped particle’s thermal motion resulted in a 6D stiffness matrix by applying equipartition theorem thereby calibrating the system. Such complexity is necessary if we consider that torques and forces also affect the motion in all three dimensions.

The precise control of 3D microscopic particles by optical tweezers allows for the assembly of multipart micromechanical structures. This was demonstrated in the works of Rodrigo and coworkers [90,91] in creating planar, and three-dimension-al assemblies of shape-complementary structures. In the 3D approach, they used a feature-rich microelement system coupled with latching mechanisms to lump robust structures together that could remain stable even after turning off the trapping beams (assembling composite structures by light). In this proof-of-concept demonstration

¯

¯˙=R−dU(¯)d¯+∆M

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two types of microcomponents were fabricated with complementary shape-features that enable joining them together to form hierarchical microstructures. One was a 13 µm long microdumbbell containing 3.8 µm diameter spherical endings, con-nected by a rod of 2.2 µm diameter. The dumbbells were designed to attach onto a complementary microblock (8 µm thickness and 17.5 µm side length). This was ac-complished by fitting the dumbbell’s spherical endings onto the circular holes (5 µm diameter) on the sides of the microblock. The assembly of the two structures required 3D micromanipulation since the dumbbell spheres cannot be inserted from the sides simply by lateral motion but they must be maneuvered from the top to achieve a latching effect. Another interesting aspect of these works was that they did not use holographic optical tweezers, but a workstation [92], consisting of a CW laser beam (λ = 1064 nm) transformed into two sets of real-time user-adjustable top-hat beams that are coupled through facing microscope objectives (50×, NA = 0.55, Olympus) into a sample chamber from opposite sides to work as counter-propagating beam traps. The relatively low NA and long working distance of the objectives allow for transporting the trapped objects in relatively large distances, even millimeters.

The same trapping setup was used in an experiment where Palima and his col-leagues showed how a two-photon polymerized microtool can enhance fluorescence microscopy [93]. Waveguides can deliver light to specific, hard-to-reach locations with micrometer precision as well as collect light for detection (e.g., endoscopy). In this work portable, free-standing waveguides are introduced that are pointed toward any desired position by the optical traps in 3D and can deliver fluorescent excitation light. The specific goal was to selectively illuminate a localized vertical target area with the fluorescence excitation beam that propagates downward from a vertically oriented microscope objective. Such target object was selected, where the direct il-lumination with vertical excitation beam is not possible. The portable microtool was designed such that it contains the functional bent waveguide part, four holder spheres that interact with the trapping beam, and their connector rods (Fig. 6.6). The bent

FIGURE 6.6 Electron Microscopic Image Highlighting the Most Important Parts of the Mobile Light Guide Used by Palima and Coworkers [93]

182 CHAPTER 6 Complex polymer microtools

waveguide part serves two functions: (1) redirect the excitation light sideways to the target and (2) modify the beam characteristics in situ, for example, produces a more tightly confined beam at its tip as compared to the incoming beam. The bending radius was 8 µm which, considering the refractive indices of the material (1.59) and that of the surrounding water (1.33), was sufficiently large to avoid the escape of the light from the waveguide due to imperfect internal reflection. The in-coupling of the excitation beam into the waveguides was assisted by a broader “light funnel.”

It was demonstrated, that the in-coupled light passed through the waveguide as planned, and it emerged at the exit. So much so that it could excite fluorescence when illuminating the fluorophore in the surrounding medium. The structures enabled the use of relatively broad beams in the focal volume of low-NA objectives by redirecting them as sharper intensity peaks that can be steered to provide localized optical illumi-nation. To demonstrate this, the waveguides were oriented toward hard fluorescence objects: vertically packed, 3 µm fluorescent polystyrene beads were selectively illu-minated with the sideway-steered beam successfully. The beads could not have been reached in a one-by-one manner with any vertical beam. It was also demonstrated that the structures can guide optical waves in the reverse direction: when light enters the tip, it is redirected toward the top-view microscope. In this experiment light was coupled into the first trapped waveguide at the “light funnel” generating a local field at its tip and then an optically manipulated second waveguide could sense this field. It showed that the waveguide can redirect localized field, which propagates off-axis beyond the microscope objective’s acceptance angle toward the low-NA top-view observation.

2.2 FUNCTIONALIZED STRUCTURESSo far the applications of “bare” optically manipulated microstructures were discussed, but the functionalization of their material or surface may further broaden their appli-cability. The polymer material of the structures is an excellent chemical platform to at-tach small functional molecules, larger biological macromolecules or even cells not to mention inorganic nanoparticles using the proper chemical and biochemical reactions. Only the imagination can limit their application: sensors, actuators, enhancers can be made of them just to name the first ones that come to one’s mind. For instance, Aek-bote and his colleagues demonstrated [94] that two-photon polymerized microstruc-tures coated with gold nanoparticles (NP) can effectively enhance fluorescence. They used 80 nm diameter gold nanospheres and coated the surface of the microstructures with them completely. Large, flat areas as well as tip-like structures with 500 nm radius of curvature were coated with the same efficiency. In all of the cases the observed fluo-rescence was enhanced by 3–6 times relative to the background, when these surfaces were brought into contact with a layer of fluorescent protein. Additionally, the local-ization of the enhancement was slightly smaller than 1 µm2. Although in this case the structures were not manipulated by light, it is possible to create complex light-steered microstructures with metal nanoparticle coating, used in a sensing application.

Vizsnyiczai and coworkers showed in an elegant work how one can combine two-photon polymerization, photoreduction of colloidal silver nanoparticles, optical trapping and Raman detection [95]. As we see, here light is used in each step of the

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preparation of the elements and in carrying out the experiment. It was demonstrated earlier that optically trapped, silver-coated microbeads can be used for localized drug detection in the outer cell membrane [96]. These spherical surface-enhanced Raman spectroscopy (SERS) probes can be useful in a series of experiments, but there are some limitations connected to their applications. First, working with spherical shapes necessitates spatial overlap of the trapping and Raman excitation laser beams, which can cause unwanted heating of the metal nanoparticles leading to the degradation of the studied molecules. Second, for successful trapping transparency of the spheres is needed and it limits the admissible extent of metal coverage. These problems were eliminated using the introduced microstructures.

The structures are developed toward such optically trappable SERS sensors, where the trapping light is spatially separated from the Raman excitation overcom-ing both of the earlier mentioned problems. In their design the microstructure is composed of three connected spheres, two of them are used to trap the structure in a dual-beam optical trap, and the third one is coated with a SERS-active metal NP layer (Fig. 6.7A). An additional feature of the microstructure preparation is that a protective shield was made above the two, trappable spheres thereby reduc-ing the possibilities for the metal NPs to attach to them. After the microtools were prepared on their glass substrate, the whole sample was immersed into aqueous silver nitrate solution, and their tip part illuminated selectively with a green laser beam. Therefore silver NPs deposited on the third sphere of the structure giving rise to a well-controlled NP density (Fig. 6.7B–C). The idea of making SERS-active silver nanostructures by photoreduction has been applied before to deposit silver nanoparticles onto glass substrates [97] or silica nanoparticles [98] and used for SERS detection.

In the experiments a time-shared, two spot optical tweezers was used, which was combined with Raman excitation and detection. The system was tested by measuring SERS spectra of the aqueous solution of emodin, an anticancer drug (1 × 10−6 M), and hypericin, a drug being considered for possible use in photodynamic therapy (1 × 10−5 M). The excitation laser power was a gentle 0.02 mW (488 nm, emodin) and 0.006 mW (532 nm, hypericin). As expected, Raman signal was only detectable over those area of the structures which was coated with silver nanoparticles, and was not when the uncoated part were measured, showing the importance of plasmonic effects for the molecules adsorbed to the silver NP surface (Fig. 6.7D).The observed detection sensitivity was found to be lower for hypericin, which could be partially due to its reduced adsorption affinity to the metal NP surfaces, which is needed to efficiently detect the molecule’s SERS signal [99]. It was also found that the intensity of the emodin SERS signal at 1667 cm−1 depended weakly on the amount of silver on the tips of the structures, in spite of significant differences in Ag layer charac-teristics (grain diameter and surface coverage). This was possibly due to exceeding the optimal nanoparticle diameter for maximal SERS enhancement at the high Ag density layers [100], the effect of which was compensated by the increased surface coverage. As a summary, the presented optically maneuverable, tailor-made SERS microprobes, after being equipped with the desired tip geometry can be applied in microfluidic environment for targeted and sensitive SERS measurements.

184 CHAPTER 6 Complex polymer microtools

Optical manipulation of cells has already resulted in a wealth of information on their mechanical characteristics [19], growth [101], fusion [102] or on their 3D struc-ture using fluorescent microscopy [103]. Trapping and relocation of cell organelles has also been demonstrated [15,104,105] and used for microrheology measurements [18]. However, when high NA objective focuses the intense trapping beam into the cells, it can be potentially dangerous for the cell, as studied on various systems [106–108]. Microbeads have already been used as intermediate objects to reduce the direct irradiation of the cells to lessen these effects [109]. Microbeads, on the other hand are limited to motions only with three degrees of freedom and still bring the trapping field close to the trapped object. To overcome these limitations, the use of extended, specifically designed intermediate objects is a promising alternative.

Aekbote and coworkers produced a microtool that can be used for indirect opti-cal trapping of fluid-borne single cells with the possibility of six degrees of freedom

FIGURE 6.7

Optical microscopy image of four SERS structure (A) and scanning electron microscopic (B, C) images of the optically maneuverable SERS platform. The silver NP layer deposited onto the glass and their tip spheres appears as a dark stripe across the optical image (A). SERS spectra of emodin dissolved in the solution surrounding the microstructures measured on two, slightly different structures (D). The two (lower) smooth lines are Raman spectra that were measured off the Ag NPs.

Reprinted with permission from Vizsnyiczai et al. Langmuir 2015;31:10087–10093 [95]. Copyright (2015)

American Chemical Society.

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motion [44]. The concept of the microtool itself, shown on Fig. 6.8A, is based on earlier designs [45,93], but has two main advantages: one is moving the trapping beam into several micrometers distance relative to the cell, and the second is that it can perform fast translational and rotational motion with up to tens of micrometer per second speed along their long axis; the actuation required a holographic optical trap with four focal spots. Another advantage of the indirectly trapped structures is that the refractive index contrast between the trapped structure (1.6) and that of water (1.33) is much higher than in the case of cell-water (cell: 1.35 [110]), allowing for more stable trapping and therefore much higher speed for translation or rota-tion (Fig. 6.8B). The applied structure consisted of four trapping spheres forming a square in a planar arrangement that are connected to a plate which was used to attach the cells to it, and also contained their connecting rods. In the presented work, the au-thors used biotinylated cells and streptavidin-coated microtools and showed that the cells attach to the structures within about only 2s but other scenarios are also possible for cell binding utilizing the extracellular polysaccharide layer of many cell lines.

FIGURE 6.8

Conceptual drawing of an indirectly trapped cell held by a microstructure that is actuated (translations and rotations) by holographic optical tweezers of 4 beams (A). Optical image sequence of an indirectly trapped cell rotated around z by the holographic trap (B). A steady reference cell is marked by asterisk; the time difference between the images is 4 s, scalebar: 10 µm.

186 CHAPTER 6 Complex polymer microtools

3 CONCLUSION AND OUTLOOKIn this chapter we surveyed the most important results on light-actuated microstruc-tures made mostly by laser-based polymerization. We show that the method of two-photon polymerization (TPP) enables the preparation of complex 3D microtools of practically any arbitrary shape. TPP is particularly important, when precise, tailor-made structures are needed for proof-of-concept experiments. Representative ex-amples for the application of the microtools were also shown that were categorized according to the various laser-based actuation methods that exploit radiation pres-sure, single or multi-beam optical tweezing or their combination. Applications of mi-crotools with various surface treatments such as metal nanoparticles or protein were also shown to emphasize their added capabilities in chemical or biological studies. In summary, TPP has proven to be an efficient tool to prepare versatile, tailor-made 3D microstructures for light actuation. Additionally, light can be used in numerous exotic ways to position, translate, rotate or even assemble these microstructures that eventually carry out a useful task. TPP is continuously improving in terms of feature size, approaching the 50 nm level from above [35] and also in terms of the micro-structure size stretching out to the millimeters regime [41]. In the field of optical manipulation the improvement is pointing toward exotic beam shapes [55] with the perspective of novel actuation schemes. The combination of these two—preparation and actuation with light—holds the promise to advance microrobotics toward micro-devices for applications in physical, biological, and medical research on the single cell or the molecular level.

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193

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00007-0Copyright © 2017 Elsevier Ltd. All rights reserved.

Takeshi Hayakawa, Hisataka Maruyama, Fumihito AraiNagoya University, Nagoya, Japan

CHAPTER OUTLINE

1 Overview of Optically Driven Micro- and Nanorobots ........................................... 1942 Multiple-Trap Manipulation System for Light Robotics ......................................... 195

2.1 Introduction ..................................................................................1952.2 System Configuration .....................................................................1962.3 Evaluation of Teleoperation System ..................................................2022.4 Summary ......................................................................................205

3 Fabrication Technology for Light Robotics .......................................................... 2063.1 Introduction ..................................................................................2063.2 Concept of 3D Hybrid Nanorobot Integrating Functional

Nanomaterials ...............................................................................2063.3 System Configuration .....................................................................2073.4 Fabrication of the Hybrid Nanorobot ................................................2083.5 Manipulation of the Hybrid Nanorobot .............................................2153.6 Summary ......................................................................................218

4 Sensing Technology for Light Robotics ............................................................... 2204.1 Introduction ..................................................................................2204.2 Concept of Optical Multisensing Microrobot ......................................2214.3 Materials and Method .....................................................................2224.4 Evaluation Results of the Optical Multisensing Microrobot .................2234.5 Summary ......................................................................................228

5 Biomedical Applications of Light Robotics .......................................................... 2295.1 Introduction ..................................................................................2295.2 Measurement of Contact Force of Microrobot to Red Blood Cells ........2295.3 Single Cell Puncture by Using Laser Heating of CNT .........................2295.4 Measurement of pH on Virus-Infected Cell Membrane .......................2315.5 Summary ......................................................................................233

References .............................................................................................................. 234

Optically driven micro- and nanorobots 7

194 CHAPTER 7 Optically driven micro- and nanorobots

1 OVERVIEW OF OPTICALLY DRIVEN MICRO- AND NANOROBOTSMicromanipulation is one of the key techniques in recent biotechnology. There have been a lot of studies on micromanipulations based on electric, magnetic, fluidic, acoustic, or optical force. Among these manipulation techniques, optical microma-nipulation, which is also known as “optical tweezers” or “laser tweezers,” have been widely used from the first report by Ashkin [1–3]. Optical tweezers offer many advan-tages for micromanipulation in biotechnology, such as high positioning accuracy and low risk of contamination. Furthermore, little modification is needed to install optical tweezers on commercial microscopes. Therefore, there are a lot of studies on biologi-cal applications using cell manipulation based on optical tweezers [4,5].

Despite the advantages of optical tweezers, there are concerns about possible cell damage, such as optical or thermal damage, when we directly irradiate biological cells with lasers. Furthermore, most early studies using direct irradiation have been mostly limited to the transportation or positioning of single cells. Therefore, in 2000, we proposed indirect manipulation using optically driven micro- and nanorobots in order to achieve a variety of minimally-invasive cell manipulation possibilities [6]. An updated visualization of this concept is shown in Fig. 7.1. The robot can be used in cell culture dish or microfluidic chip to provide stable and highly control-lable environment and low risk of contamination. These platforms can be set on the usual microscope to enable brightfield or fluorescent observation of biological cells. Thus, the optically-manipulated micro- and nanorobots can be used by putting the microfluidic chip or culture well on a microscope that has been modified to install an optical micromanipulation system.

Using optically driven micro- and nanorobots achieves trapping or transporta-tion of cells with less damage compared to directly irradiated cells as depicted in Fig 7.2A,B. Furthermore, attitude or orientation control of the cells and force appli-cation to cells can be realized using multiple manipulated microrobots, as shown in Fig 7.2C,D. Other types of cell manipulations can be realized by using appropriately

FIGURE 7.1 Concept for Optically Driven Micro- and Nanorobots

1952 Multiple-trap manipulation system for light robotics

functionalized robots. For example, measurement of extracellular and intracellular environment, such as pH, temperature, or ionic concentration is realized with micro-robots that are dyed with environment-sensitive fluorescent reagents [7]. Moreover, cell surgery, such as puncture or injection into the cells can be achieved by utilizing functional nanomaterials, such as carbon nanotubes (CNTs) or nanoparticles [8].

To date, we have succeeded in various cell manipulations, such as transporta-tion, pH and temperature measurement, and cell puncture with proposed optically driven micro- and nanorobots. In this chapter, we introduce recent studies about this research. In Section 2, we explain high-speed multiple-trap manipulation system with holographic optical tweezers, which is an important technology for manipulat-ing optically-driven micro- and nanorobots [9]. In Section 3, we introduce fabrica-tion method of three-dimensional (3D) hybrid nanorobots which has hybrid structure of robots and functional nanomaterials [8]. In Section 4, we explain a method for measuring multiple parameters in extracellular/intracellular environment with fluorescent-dyed microrobots [10]. Finally, in Section 5, we introduce biological applications of these robots (Fig. 7.2).

2 MULTIPLE-TRAP MANIPULATION SYSTEM FOR LIGHT ROBOTICS2.1 INTRODUCTIONIntroducing a single laser beam into a microscope can trap only one object and only transportation or positioning can be achieved with a single trap. By extending the

FIGURE 7.2 Various Cell Manipulations With Optically-Driven Micro- and Nanorobots

(A) Trap or transportation (B) Trap or transportation (C) Attitude and orientation control (D) Force application (E) Extracellular/Intracellular measurement (F) Cell surgery.

196 CHAPTER 7 Optically driven micro- and nanorobots

optical tweezers system from a single trap to multiple traps, various applications are realized, as shown in Fig. 7.3. Time-shared scanning (TSS) method [11,12], general-ized phase contrast (GPC) method [13], and computer-generated hologram (CGH) method [14] are well-known multibeam generation methods. In the present study, our group focused on holographic optical tweezers (HOT) using the CGH method, which can be extended to three-dimensional manipulation. However, the manipula-bility of HOT was greatly limited by the speed of the beam control. This was mainly due to the computation time for generating the hologram. Recent progress in parallel computing using the graphics processing unit (GPU) contributed to a breakthrough regarding the problem [15–17]. Since the calculation of optics and photonics exhibits parallelism, parallel computing is suitable to compute optics and photonics phenom-enon numerically. This real-time generation of holograms enables the mass manipu-lation of small objects in real-time via HOT. Thus the position information inputted by an operator immediately influences the changes of position or status of the objects in the microworld.

Additionally, the user interface of optical tweezers system is also important to realize dexterous manipulations, such as shown in Fig. 7.3. Our group has proposed bilateral teleoperation system of optical tweezers to achieve such cell manipula-tions [18–20]. This bilateral teleoperation technique is also integrated to the GPU-accelerated HOT system.

2.2 SYSTEM CONFIGURATION2.2.1 ConceptIn this section, we describe a bilateral teleoperation system that is capable of multiple manipulations and multiple force feedback. The proposed system is composed of a double-arm master-slave system, which uses HOT accelerated by a GPU. This system is controlled with a force reflection type bilateral master-slave control technique [21]. The masters are two haptic devices (Sensable PHANTOM Omni), and the slaves are two laser-trapped microbeads made of polystyrene (diameter: 3 µm, refractive index: 1.59). A schematic diagram of the proposed system is shown in Fig. 7.4. The

FIGURE 7.3 Applications of Multiple Point Manipulation With Optical Tweezers

(A) Parallel manipulation (B) Force application (C) 3D manipulation.

1972 Multiple-trap manipulation system for light robotics

positions of the microbeads are controlled along the trajectories of the haptic devices, and the forces to which the slaves are subjected, are measured and fed back to the haptic devices. In this system, HOT acts as an interface between the operator and the microworld for telexistence using input devices, such as computer mouse or keyboard [22–24]. Therefore, it is important to feedback the forces to which the manipulated objects are subjected. Chapter 1 discusses several types of user interfaces.

2.2.2 Optical systemThe optical system of HOT is shown in Fig. 7.5. An infrared laser source (Yb fiber laser, IPG Photonics) having a wavelength of 1064 nm is used and introduced to a neutral density filter (ND) to adjust the laser power. A polarizer (PL) is placed for adjusting the polarization plane to match the aligned nematic liquid crystal display

FIGURE 7.4 Schematic Diagram of the Teleoperation System Using Holographic Optical Tweezers

The coordinate systems of the master, slave, camera, and hologram are denoted as Om, Os, Oc, and Oh, respectively [9].

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2012;20(4):3633–3641.

198 CHAPTER 7 Optically driven micro- and nanorobots

of a spatial light modulator. A beam expander (L3, L4) expands the diameter of a laser beam so that the display of a spatial light modulator is irradiated with a laser beam as large as possible. An iris diaphragm (Iris) resizes the laser beam diameter. The collimated polarized laser beam is introduced to a spatial light modulator (SLM: Hamamatsu Photonics X10468-03, frame rate: 60 Hz) [25], which modulates the two-dimensional phase distribution of the beam based on the displayed Kinoform hologram. The displayed hologram is transferred to the back focal plane of an objec-tive lens (OL: Olympus UPLSAPO 100XO, 100x, N.A.: 1.40) via relay lenses (L1, L2) and dichroic mirror (DM). The relay lenses scale down the diameter of a laser beam for increasing the max spatial frequency of the modulated laser beam. This process increases the maximum diffraction angle and expands the working space on the focal plane of the OL where multiple laser spots are generated. The target objects are trapped at the laser spots.

Holographic optical tweezers have a problem of generating ghost traps or distur-bances (i.e., disturbances of control system), such as one caused by the zero-order beam, which is the nondiffracted beam from the SLM. The power of the zero-order beam can increase during dynamic control when the diffraction efficiency on the SLM decreases. The reduction of diffraction efficiency depends on the response of the liquid crystal of the SLM. Therefore, the zero-order beam should be removed for stable and high-speed manipulation. Here, an optical system for filtering the zero-order beam is implemented [26]. A composite hologram φ′h which is composed of a Fourier hologram φh and a Fresnel phase lens φlens axially separates the zero-order beam from the first-order beam as shown in Fig. 7.6, and calculated as follows:

φ φ φ π′ = +( )mod 2h h lens (7.1)

φ′h

φ′h=(φh+φlens)mod2π

FIGURE 7.5 Optical System of Holographic Optical Tweezers [9]

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2012;20(4):3633–3641.

1992 Multiple-trap manipulation system for light robotics

A Fresnel lens of concave type is calculated as follows [27]:

φ πλ

π= r

fmod 2lens

2

(7.2)

Here, r is the radius from the center of the hologram (i.e., the distance from an optical axis); λ is the wavelength of a laser beam; f is the desired focal length of a Fresnel phase lens. Furthermore, L3 and L4 are placed not to be a conjugate relation; the distance between these lenses is chosen to focus the zero-order beam at the spa-tial filter (SPF). This SPF contains a small circular disc made of Cr/Au coating that reflects and removes the focused zero-order beam. On the other hand, the diffracted

φlens=πr2λfmod2π

FIGURE 7.6 The Phase Distribution of Centered Hologram is an Example of Composite Hologram by Using a Fresnel Phase Lens Shown in Left Side and a Fourier Hologram Shown in Right Side for Separating a Zero-Order Beam and a First-Order Beam [9]

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2012;20(4):3633–3641.

200 CHAPTER 7 Optically driven micro- and nanorobots

beam is almost undisturbed and generates multibeam at the Fourier plane with this optical system. The efficiency of this filtering system was experimentally confirmed. In this optical system, the designed parameter of maximum workspace diameter and resolution was 160 and 0.3 µm. In fact, workspace diameter is more than 160 µm, and positioning resolution is 0.2 µm at the central region, and these results were experimentally confirmed.

2.2.3 Control and force feedback systemThe block diagram of a bilateral control system is shown in Fig. 7.7. Using camera images, the master position vector Xm is transformed to position Xcm in the camera coordinates system (common coordinate) using Tpmc. The positions of masters and slaves are managed in this common coordinate system. Then, Xcm is transformed to Xsm in the slave coordinate system with Tpcs. The CGH generator calculates holo-grams φ (sampling size: 512 × 512) using a GPU (NVIDIA Tesla C1060) in real time. The Gerchberg-Saxton method is used to calculate the holograms [28]. The processing rate of a GPU is 250 Hz and 30 times faster than the rate (8 Hz) of a central processing unit (Intel Core i7 975). Using the optical system, laser spots are generated at Xsi and slaves settle at Xs in the slave coordinate system.

Here, we propose a force measurement method using the analysis of microscopic camera images shown in Fig. 7.8. If the trap stiffness parameter Kt is known, then the forces to which slaves are subjected can be estimated as follows [29]:

= −F K X X( )s t s si (7.3)

where Kt is assumed as a constant in space-time and is measured by the drag force method [30]. Chapter 1 discusses various calibration methods for simple micro-spheres and Chapter 3 discusses the rigorous calibration of arbitrary objects. For measuring Kt, we use two laser-trapped beads, the total laser power of which are the same as that of the experimental condition listed in Table 7.1. Microscopic images are acquired by a high-speed CCD camera (Point Grey Research Grasshopper GRAS-03K2C, pixel resolution: 640 × 480, frame rate: 200 fps). Xs is transformed to Xcs by analyzing images obtained from the CCD camera. We use the normalized cross cor-relation method for detecting Xcs. Ideally, we would like to determine the positions of

Fs=Kt(Xs−Xsi)

FIGURE 7.7 Block Diagram of Bilateral Control System [9]

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2012;20(4):3633–3641.

2012 Multiple-trap manipulation system for light robotics

the multiple beams in the common coordinates Xcsi with the CCD camera. However, the laser positions are invisible because the lasers are scattered or occluded by slaves or other objects. Therefore, estimation of Xcsi is important for positioning and force sensing.

Here, we employ a simple way of using the position of the master Xcm for mea-suring the slave forces. Using Xcm, Xcs, and the unit exchange factor (from the com-mon coordinates to the slave coordinates) Kpcs, we can estimate Fs without Xcsi as follows:

= −F K K X X( )s t pcs cs cm (7.4)

=K K Ks t pcs (7.5)

Fs=KtKpcs(Xcs−Xcm)

Ks=KtKpcs

FIGURE 7.8 Schematic Diagram of the Force Measurement by Using the Image Analysis of Microscopic Camera Images [9]

Copyright 2012 Optical Society of America. Reprinted with permission from Onda K, Arai F. Opt Express

2012;20(4):3633–3641.

Table 7.1 Experimental Condition for the Evaluation of Bilateral Teleoperation

Temperature 293 K

Total laser power (OL) 20 mWOptical trap stiffness Kt 11 pN/µm/beamControl cycle 100 HzCGH generation cycle 250 HzForce feedback gain Sf 1010

202 CHAPTER 7 Optically driven micro- and nanorobots

The detected forces are on the order of pN and are too small to sense using haptic devices. Therefore, we need to amplify the detected forces using force feedback gain sf. The amplified forces Fs (on the order of milliNewton) are fed back to the masters. The refresh rate of the forces is approximately 100 Hz because of the limitation of the control system. Therefore, the proposed system synchronizes each control element at 100 Hz, except for the SLM. Ideally, the refresh rate must be 1 kHz for continuous force feedback [31]. The manipulation workspace is limited by the field of view of the microscope, and we limited the workspace of this experiment to 23 × 17 µm to increase the resolution of force sensing, which depends on the ratio by CCD camera pixels to observed area. This space is inside the laser movable area, which is decided according to the maximum spatial frequency of the SLM (25 lp/mm) [32]. And the positioning resolution is 0.2 µm, which is evaluated experimentally. This resolution is decided based on the calculation size of the hologram.

2.3 EVALUATION OF TELEOPERATION SYSTEMFor the evaluation of bilateral teleoperation system, the manipulation of slaves was done in water. The experimental conditions are shown in Table 7.1 and the experi-mental results are shown in Fig. 7.9. Fig. 7.9 shows the clockwise operation of the two masters and clockwise rotation of the two slaves. In the slave movement, the

FIGURE 7.9 Images of the Teleoperation of Clockwise Rotation

Upper images show the operation of masters and lower show the movement of slaves which moves along the trajectory of masters.

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2012;20(4):3633–3641.

2032 Multiple-trap manipulation system for light robotics

cross markers indicate the target positions of multibeam, Xcm, and the squares indi-cate the slave positions Xcs. These markers are processed and overlayed on the CCD camera images in real time. The square tracked each slave smoothly, and the detec-tion of Xcs using image analysis was successful. The crosses (Xcm) approximately agreed with the squares (Xcs) under static condition (at 0 ms in Fig. 7.9). This is because this system uses the equality of Xcm and Xcs of a steadily trapped bead under static condition for positioning calibration, and then Tpcs is decided as following lin-ear map:

=X T T Xcs psc pcs cm (7.6)

=T H H Spcs trans rot pcs (7.7)

Here, Tpcs is coordinate transformation from slave coordinate to common coordi-nate by using camera system shown in Fig. 7.7, and this parameter is a constant that depends on the hardware. Htrans and Hrot shows the coordinate transformation about the translation (offset) and the rotation, and spcs is the motion ratio between the com-mon coordinate and the slave coordinate. Tpcs is necessary to compensate the differ-ence between slave coordinate system and common coordinate system.

Fig. 7.10 shows the time response of the position and the force associated with the left-side master-slave. With respect to the position response, the slave position follows the master position with a time delay (Fig. 7.10A). And the measured slave force indicates the tendency of Stokes drag compared with estimated Stokes drag calculated with the slave velocity (Fig. 7.10B). But the slave force is approximately 20 times stronger than Stokes drag. This problem mainly comes from the time-delay and the deviation of multibeam control. The slave forces can be measured by using the difference between slave positions Xcs and laser positions Xcsi. Ideally, we should use these parameters, but Xcsi cannot be observed. Therefore, the position instruction Xcm was used as substitute for Xcsi. But Xcm is not equal to Xcsi because Xcsi has the time-delay and the deviation against Xcm.

As for time-delay, ramp response of Fig. 7.11 shows the time-delay Td is 70 ms, and it shows the large error between Xcsi and Xcm. It is interesting that Td is almost the same as the response time of the liquid crystal of SLM (response of the liquid crystal: rise 30 ms, fall 80 ms). Therefore, we proposed the modified slave forces fs which compensated for the time-delay. If the time delay Td is assumed to be 70 ms and constant, the modified slave force fs is indicated as follows by using this relation (Xcsi(t) = Xcsi (t − Td): Td = 0.07 s):

( )= − −f t K X t X t T( ) ( ) ( )s s cs cm d (7.8)

The modified slave force which was calculated by applying this modification to previous experimental data is shown in Fig. 7.10C. The difference between modified slave force and Stokes drag is smaller than that between the unmodified slave force and Stokes drag (Fig. 7.10B), and shows the tendency of Stokes drag. This modified slave force has time-delay but the accuracy of slave forces was improved.

Xcs=TpscTpcsXcm

Tpcs=HtransHrotSpcs

fs(t)=KsXcs(t)−Xcm(t−Td)

20

4C

HA

PTE

R 7

Optically driven m

icro- and nanorobots

FIGURE 7.10 Results of Bilateral Teleoperation of the Left-Side Master-Slave

In the force graphs, right-side y-axis shows estimated Stokes drag calculated from the slave velocity. (A) Position response. (B) Left-side y-axis is slave force Fs and right-side is Stokes drag. (C) Left-side y-axis is modified slave force fs and right-side is Stokes drag.

2052 Multiple-trap manipulation system for light robotics

As for deviation, the static condition at 0 ms of Fig. 7.10A shows the static devia-tion of the slave position against the master position. This deviation affects the error of feedback forces, such as the force in the static condition at 0 ms of Fig. 7.10C. This deviation comes from the positioning resolution limit and the calibration error. The positioning resolution of this system is 0.2 µm and 13% of the radius of a bead (diameter: 3 µm). We think the tendency of slave forces is obtained regardless of the error, but this error should be compensated for accurate force measurements. On the other hand, the calibration was done by one steadily trapped bead, and Eqs. (7.6) and (7.7) were used for coordinate transformation. And in this calibration, we used two positioning points of a trapped bead because of two-dimensional manipulation. But the deviation occurred. It is thought that there are two reasons. First, the positioning resolution limit affects the calibration error. Second, the linear approximation of this calibration is not sufficient and we need higher approximation. This problem about positioning deviation will be tackled in our future works.

2.4 SUMMARYIn this section, we demonstrated multibeam bilateral teleoperation of holographic optical tweezers accelerated by a GPU. This system can feedback two forces to which two slaves are subjected. The property of this method is multitrapping and

FIGURE 7.11 Ramp Response of a Laser Beam and a Slave Against Position Instruction of a Ramp Input in Trajectory Control

Xcsi was measured by observing a laser beam which was reflected by a mirror set up on the plane of OL.

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2012;20(4):3633–3641.

206 CHAPTER 7 Optically driven micro- and nanorobots

multiforce-sensing. Therefore, it is possible to squeeze or strain microscale objects dexterously with measuring forces at multiple points. We discussed the methodology and the problem associated with the accuracy and the response of multibeam control. Especially, the response time is big problem in high speed manipulation using the GPU acceleration because it causes the error of force sensing. And we proposed the solution of this problem by accepting time-delay of force feedback. The constructed system reveals the possibility of telexistence between the operator and the micro-world, which means that it is possible to analyze the mechanical characteristic of living cells in real time.

3 FABRICATION TECHNOLOGY FOR LIGHT ROBOTICS3.1 INTRODUCTIONAs a microtool manipulated by using optical tweezers, transparent microbeads, such as polystyrene microbeads are commonly used. However, only transporta-tion, positioning, and application of force are achieved with microbeads of simple spherical shape. Thus, we have proposed fabrication method of optically driven micro- and nanorobots based on thermo responsive gel material, photolithogra-phy technique, or self-assembly technique [33,34]. Although these fabrication methods enable us to fabricate robots with spherical or two-dimensional shape, it is difficult to fabricate 3D shape. In this section, we explain the fabrication method of micro- and nanorobots with 3D shape based on femtosecond laser expo-sure [35,36]. Furthermore, we introduce a concept of hybrid nanorobot having functional nanomaterials [8].

3.2 CONCEPT OF 3D HYBRID NANOROBOT INTEGRATING FUNCTIONAL NANOMATERIALSToward single-cell manipulations or surgeries on small cells, we proposed an opti-cally driven hybrid nanorobot that has structures of nanometer order and a probe that integrates functional nanomaterials. In order to fabricate the optically-driven micro- and nanorobots with 3D shapes, we employed a femtosecond laser expo-sure that can fabricate a 3D fine structure of order ≈100 nm [35,36]. The fabricated nanorobots having nanometer-order structures can be applied to single small cells with the fine structure and high positioning accuracy of optical tweezers. More-over, our lithography technique allows us to fabricate the hybrid structure of the photoresist and functional nanomaterials, as shown in Fig. 7.12. By impregnat-ing functional nanomaterials into the nanorobot, it becomes possible to create a new function for the nanorobot that was previously impossible because of the small manipulation force of optical tweezers. In this study, we succeeded in fab-ricating a hybrid nanorobot integrating carbon nanotubes (CNTs) that have high photothermal efficiency [37].

2073 Fabrication technology for light robotics

3.3 SYSTEM CONFIGURATIONThe experimental system consisted of a femtosecond laser exposure system for the fabrication of the nanorobot and a HOT system for nanorobot manipulation. Both systems were integrated in the same microscope, as shown in Fig. 7.13. We have

FIGURE 7.12 Concept of the Hybrid Nanorobot With a Functional Nanomaterial [8]

Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

FIGURE 7.13 System Schematic of Femtosecond Laser Exposure and the HOT System [8]

Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

208 CHAPTER 7 Optically driven micro- and nanorobots

constructed the femtosecond laser exposure system to fabricate the nanorobot. As the laser source of the exposure system, an ultrashort pulsed titanium–sapphire laser (Chameleon, Coherent Co. Ltd.) with a wavelength of 780 nm and pulse width of 140 fs was employed. The laser beam was focused on the sample through a micro-scope objective lens (Olympus Corporation, UPLSAPO 100XO). We employed a piezoelectric XYZ stage (Physik Instrumente GmbH Co., P-563) for positioning the exposed samples. The exposure area of this system depends on the working dis-tance of the objective lens and the range of motion of the positioning stage. The exposure area of the system was 300 × 300 µm in the horizontal plane and 130 µm in the vertical axis. For the manipulation of nanorobots, we employed the HOT system [9] which is explained in Section 2. Using1064-nm NIR wavelength causes rela-tively less damage to the living cells and the HOT system can independently control multiple trapping beams.

3.4 FABRICATION OF THE HYBRID NANOROBOT3.4.1 Fabrication processWe realized the proposed hybrid structure of the nanorobot by sandwiching the CNTs in negative photoresist layers. We used a negative photoresist (Nippon Kayaku Co. Ltd., SU-8) as the material of the nanorobot. The fabrication process of the hybrid nanorobot is shown in Fig. 7.14 and can be outlined as follows:

1. Spin coat SU-8 on a glass substrate to a layer thickness of 7 µm.2. Coat and dry the CNTs (multiwalled CNTs, Sigma-Aldrich Co. LLC.)

dispersion on the spin-coated SU-8 layer.3. Spin coat a second SU-8 layer over the CNTs.4. Pattern SU-8 by femtosecond laser exposure.5. Develop the nanorobot.6. Package the chip in polydimethylsiloxane (PDMS).

With this process, we can integrate a CNT into the nanorobot and use it as a manipulation or surgery probe. The materials to be integrated are not restricted to CNTs but can also include various nanomaterials. For example, lithium niobate nanowires optimized for waveguiding and second harmonic generation, as discussed in Chapter 8, would be particularly interesting for other applications.

3.4.2 Basic evaluation of the fabricationWe evaluated the processing resolution of the femtosecond laser exposure sys-tem. The fabrication parameters include laser wavelength, laser power, and scan-ning velocity of the stage. In this study, we fixed the laser wavelength and power to 780 nm and 1.0 mW, respectively. First, we evaluated the processing resolution of the exposure with SU-8 only. To evaluate the fabrication resolution, we exposed a single line and measured the linewidth and thickness with a scanning electron micro-scope (SEM). We varied the scanning velocity of the stage from 0.5 to 30.0 µm/s.

2093 Fabrication technology for light robotics

The acquired SEM images are shown in Fig. 7.15. When the lines were scanned at 30.0 µm/s, the exposed line was broken at development. The cause of the breakage is thought to be the liquid bridge force activated between the exposed line and sub-strate. Therefore, as shown in Fig. 7.15, the minimum linewidth was approximately 270 nm, and the thickness was approximately 600 nm. Additionally, the thickness of the line was approximately 2 times larger than its width. This occurred because the exposed area is related to the size of the beam spot and the focal depth [38]. Each value is calculated as follows.

λ= =r

N ABeam spot:2

1.22

. .680(nm)s (7.9)

λ= =z

n

N AFocal depth:2

2

. .1190(nm)R (7.10)

For this calculation, wavelength λ = 780 [nm], numerical aperture N.A = 1.4, and refraction index n = 1.515 for the oil immersion lens are used. This ratio rs/zR = 1.75 is close to 2, the ratio of the exposed linewidth and thickness. Therefore, the line thickness became larger than the linewidth because of the shape of the beam spot

Beam spot: 2rs=1.22λN.A.=680 (nm)

Focal depth: 2zR=2nλN.A.=1190 (nm)

FIGURE 7.14 Fabrication Process of the Hybrid Nanorobot [8]

Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

210 CHAPTER 7 Optically driven micro- and nanorobots

and focal depth of the laser. Second, we evaluated the fabrication resolution when the CNTs were embedded in SU-8. We exposed single lines that include single CNTs and evaluated the linewidth and thickness in the same manner as the SU-8 case. The acquired SEM images are shown in Fig. 7.16. The linewidth and thickness of the exposure with the CNT are approximately 2 times larger than the case of SU-8 only. For example, the linewidth is 1.3 µm and thickness is 2.3 µm in the case of exposure with the CNT, and the linewidth is 0.6 µm and thickness is 1.2 µm in the case of exposure without the CNT when scanned at 15 µm/s. A cause of this phenomenon is thought to be that proton diffusion of SU-8 is enhanced as the temperature of the CNTs increases during exposure. The CNTs absorb the laser exposure slightly and the temperature might increase. In addition, the exposed lines were broken when the line was scanned at 20.0 µm/s. Therefore, the minimum linewidth was approxi-mately 1.3 µm and thickness was approximately 2.3 µm.

FIGURE 7.15 SEM Image of Exposed Line With SU-8 Only

(A) Top view, (B) perspective view (40 degrees) [8].Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):

59–67.

2113 Fabrication technology for light robotics

We evaluated the processing resolution with five samples in both cases. The eval-uation results are summarized in Fig. 7.17. The horizontal axis shows the scanning velocity. The vertical axis of Fig. 7.17A shows the linewidth and the vertical axis of Fig. 7.17B shows the line thickness. The processing resolutions of the linewidth and thickness were 270 ± 30 nm and 600 ± 120 nm in the case of exposure with SU-8 only. The processing resolutions of the linewidth and thickness were 1.3 ± 0.1 µm and 2.3 ± 0.2 µm in the case of exposure with the CNTs.

3.4.3 Design and fabrication of the on-chip hybrid nanorobotWe designed and fabricated the hybrid nanorobot by using femtosecond laser expo-sure, evaluated in the previous subsection. The design requirements for the nanoro-bot are the following:

1. It should have a CNT integrated on it.2. It should be arbitrarily manipulated in two dimensions.3. It should be stable manipulated with optical tweezers.

FIGURE 7.16 SEM Image of Exposed Line With SU-8 and CNT

(A) Top view, (B) perspective view (40 degrees) [8].Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

212 CHAPTER 7 Optically driven micro- and nanorobots

To meet these requirements, we designed the nanorobot as shown in Fig. 7.18. Although the CNT can be integrated at an arbitrary position on the nanorobot, as described in the previous subsection, the integration point must be separated from the laser trap points. If the CNT is included at a laser trap point, unintended heating of the CNT occurs, along with a significant decrease in the manipulation force, because of absorption and scattering of the laser on the CNT. Considering this factor and requirements 1 and 2, we employed three spherical trap points connected by three beams joined at the integration point of the CNT.

Second, we determined the size of the nanorobot by considering manipulation stability (requirement 3). In a microscopic environment, such as a microchannel on a microfluidic chip, physical phenomena are dominated by viscosity, in contrast with a macroscopic environment, which is dominated by inertia. Therefore, the design of the nanorobot must consider the effect of viscosity. Our group previously studied the stability of optical manipulation of a spherical target by assuming that the inertia fac-tor and random thermal force are negligible [11]. In this study, we considered only spherical trap points for simplicity; therefore, the equation of motion can be written as

πη + =r x Kx6 0t (7.11)6πηrtx˙+Kx=0

FIGURE 7.17 Relationships Among Scanning Velocity, Exposed Line Width, and Line Thickness [8]

(A) Line width evaluation (B) Line thickness evaluation.Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

2133 Fabrication technology for light robotics

where η is a coefficient of viscosity of a solution, rt is radius of a trap point, x is a coordinate of the laser focus center, and K is trap stiffness of the optical tweezers. Here, we assumed that trap stiffness K is constant when x is located inside the trap point; otherwise, it is zero:

= ≤K x K x a( ) (| | ) (7.12)

= >K x x a( ) 0 (| | ) (7.13)

If the laser position is at x = di at t < 0 and the position is updated to x = 0 at time t = 0 (Fig. 7.19A), the position of the trap target x at time t can be written as

πη= −

x dK

rtexp

6.i

t

(7.14)

K(x)=K⋯(|x|≤a)

K(x)=0⋯(|x|>a)

x=diexp−K6πηrtt.

FIGURE 7.18 Design of the Hybrid Nanorobot

(A) Top view. (B) Side view [8].Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):

59–67.

FIGURE 7.19 Stable Manipulation Condition

(A) Stable condition 1. (B) Stable condition 2 [8].Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):

59–67.

214 CHAPTER 7 Optically driven micro- and nanorobots

Assuming that the Brownian motion of the object is small compared with the size of the object, the settling time ts, which is the time taken to settle in the area of the Brownian motion around the goal point of each step, can be written as follows [11]:

πη=

t

r

Kd

K

k T

6ln ,is

t

B

(7.15)

where kB is the Boltzmann constant and T is the absolute temperature of a solution. In this case, each time step ∆t must be longer than the settling time ts (∆t ≥ ts), and laser position di can be written as d1 = v∆t when the manipulation laser moves at constant velocity v. Then, the velocity limitation for stable manipulation can be written as follows (condition 1):

πη≤

∆∆

vt

k T

K

K

rt

1exp

6.B

t

(7.16)

Another limitation on manipulation velocity comes from the condition that the laser focus center must be located in the trap point at each time step. Otherwise, the optical trap force does not act on the trap point, and the trap point is out of control with respect to optical manipulation. If the laser focus center is located at x = 0 at time t = 0 and moves to xi = v∆t at the next step (Fig. 7.19B), the condition of the laser focus center inside the trap point can be written as

∆ ≤v t r .t (7.17)

Therefore, another velocity limitation on stable manipulation is the following (condition 2):

≤∆

vr

t.t (7.18)

From condition 1 Eq. (7.16) and condition 2 Eq. (7.18), we plotted the relation-ship between the trap point radius and manipulation velocity, as shown in Fig. 7.20. We employed the following values for the analysis.

Control time step: ∆t = 16.7 msBoltzmann constant: KB = 1.38 × 10−23 J/KSolution temperature: T = 310 KOptical trap stiffness: K = 11 pN/µmCoefficient of viscosity: η = 6.85 × 10−4 Pa s.We determined ∆t from the communication rate of the spatial light modulator

(SLM), which is used for laser hologram generation (60 Hz). Furthermore, we employed the value of optical trap stiffness as previously evaluated [9]. From Fig. 7.20, the stable condition is around rt ≈ 2.9 µm. We experimentally con-firmed the maximum manipulation velocity of the microbeads. The evaluation

ts=6πηrtKlndiKkBT,

v≤1∆tkBTKexpK6πηrt∆t.

v∆t≤rt.

v≤rt∆t.

2153 Fabrication technology for light robotics

was performed for microbeads of radii 1.5, 2.5, and 3.5 µm. Their maximum velocities were 110, 160, and 140 µm/s, respectively. Therefore, we used rt ≈ 2.5 µm as the trap point for the nanorobot in this study. The thickness and length of the joint beams, and the diameter and height of the holding pillar were deter-mined experimentally to not be broken by the liquid bridge force during the development of the nanorobot. The thickness and length of the joint beams were 1.3 and 5.8 µm, and the diameter and height of the holding pillar were 1.5 and 7.0 µm, respectively.

Finally, we fabricated the hybrid nanorobot as shown in Fig. 7.21. Fig. 7.21 shows a picture and SEM images of the fabricated sample. From Fig. 7.21, the pro-posed hybrid nanorobot with the CNT was successfully fabricated.

3.5 MANIPULATION OF THE HYBRID NANOROBOTWe used the HOT system to manipulate the hybrid nanorobot. The optical twee-zers can manipulate the nanorobot with high spatial resolution. Therefore, the nanorobot can suitably access a single cell with optical tweezers. To introduce

FIGURE 7.20 Analysis Result of Relationship Between Trap Object Size and Manipulation Velocity [8]

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59–67.

216 CHAPTER 7 Optically driven micro- and nanorobots

the nanorobot into a workspace on a microfluidic chip, we utilized femtosecond laser ablation [39] to cut the holding pillar. After that, it was manipulated by HOT. The concept of the femtosecond laser ablation of the holding pillar is depicted in Fig. 7.22. Fig. 7.23 shows actual microscope images of the introduction and optical manipulation of the hybrid nanorobot. By ablating the holding pillar, the fabricated nanorobot was successfully introduced into the workspace on a microfluidic chip, as shown in Fig. 7.23.

FIGURE 7.21 Image of Fabricated Hybrid Nanorobot

(A) Microfluidic chip with the nanorobot. (B) SEM image of top view of the nanorobot. (C) SEM image of perspective view of the nanorobot [8].

Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):

59–67.

2173 Fabrication technology for light robotics

To evaluate optical manipulation characteristics, we evaluated the step, ramp, and frequency responses of the nanorobot. The positions of the nanorobot were mea-sured by template matching on the basis of the images acquired by the CCD camera attached to the microscope (Fig. 7.24A). We performed each evaluation in three directions, considering the symmetry of the nanorobot. The definitions of the direc-tions are shown in Fig. 7.24A. Fig. 7.24B shows the step response of the nanorobot when 2.0 µm inputs were applied at time =0.0 ms. The horizontal axis shows elapsed time, and the vertical axis shows the position of the nanorobot and the input. The step response of all the directions had a dead time element and a first-order lag ele-ment. The values are approximately 60 and 30 ms, respectively. The main cause of the dead time was the rise time (20 ms) and communication time (17 ms) of the SLM for laser hologram generation. After reaching the target position, the position of the nanorobot became stable. The position difference from the target (approxi-mately 0.1 µm) was thought to be caused by the error in the position measurement.

FIGURE 7.22 Concept of Introducing the Nanorobot by Femtosecond Laser Ablation [8]

(A) Fixed hybrid nanorobot (B) Femtosecond laser ablation (C) Released nanorobot (D) Optical manipulation of the nanorobot.

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218 CHAPTER 7 Optically driven micro- and nanorobots

Fig. 7.24C shows the ramp response of the nanorobot when 30 µm/s inputs were applied. The horizontal axis shows elapsed time, and the vertical axis shows the position of the nanorobot and the input.

Fig. 7.25 shows the gain and phase diagrams of the nanorobot when a sinusoidal input with 2.0 µm amplitude was applied. We evaluated the response at frequencies 0.1, 0.5 Hz, and every 1 Hz from 1 to 10 Hz. The horizontal axis of Fig. 7.25 shows applied frequency. The vertical axis of Fig. 7.25A shows the magnitude of amplitude of the nanorobot against the amplitude of the input. The vertical axis of Fig. 7.25B shows the phase lag time of the nanorobot against the input. These results indicate that the nanorobot can follow the input up to a few hertz in all directions. This drive frequency is thought to be sufficient for cell application.

3.6 SUMMARYIn this section, we explained a fabrication method for 3D optically manipulated nanorobot having hybrid structure with functional nanomaterials. This hybrid

FIGURE 7.23 Demonstration of the Introduction and Optical Manipulation of the Nanorobot [8]

(A) Fixed hybrid nanorobot (B) Femtosecond laser ablation (C) Released nanorobot (D) Optical manipulation of the nanorobot.Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

21

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Fabrication technology for light robotics

FIGURE 7.24 Results of the Response Evaluation

(A) Position measurement by template matching image and definition of direction. (B) Step response. (C) Ramp response [8].Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

220 CHAPTER 7 Optically driven micro- and nanorobots

structure enables the nanorobot to have new functionality by utilizing unique char-acteristics of the nanomaterials. We fabricated the proposed hybrid nanorobot with the CNT. First, we evaluated the processing resolution of the hybrid structure of the SU-8 and CNT. The processing resolutions of the linewidth and thickness were 270 ± 30 nm and 600 ± 120 nm, respectively, in the case of exposure with SU-8 only. The processing resolutions of linewidth and thickness were 1.3 ± 0.1 µm and 2.3 ± 0.2 µm in the case of exposure with the CNT, respectively. Next, we ana-lyzed the manipulation stability of HOT by considering the viscosity factor, which is dominant in the microscopic environment. From there, we determined the size of the nanorobot trap points as 2.5 µm. Then, we fabricated the hybrid nanorobot with the CNT and performed a demonstration. We performed optical manipulation of the hybrid nanorobot and evaluated its step, ramp, and frequency responses. By manipulating the hybrid nanorobot with optical tweezers, further flexible operations that target single cells become possible. The proposed hybrid nanorobot enables manipulations and surgeries of small cells. Moreover, by utilizing various charac-teristics of functional nanomaterials, a novel function can be created and integrated on optically-driven micro- and nanorobots. For example, Chapter 8 discusses lithium niobate nanowires for waveguiding and second harmonic generation.

4 SENSING TECHNOLOGY FOR LIGHT ROBOTICS4.1 INTRODUCTIONRecent researches have proven that temperature plays an important role in many cellular events, and has close relationships with cellular state and functions [40]. The pH modulates the function of many organelles and plays a pivotal role in many physiological and pathological processes [41,42]. Therefore, measurements of tem-perature and intracellular and extracellular pH can provide critical information on cell activities and the development of micro- and biocompatible sensors that can reveal these temperature and pH changes in cells has become an urgent demand.

FIGURE 7.25 Results of the Frequency Response

(A) Gain diagram and (B) phase diagram [8].Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):

59–67.

2214 Sensing technology for light robotics

Many types of microsensors for physiological cell conditions have been devel-oped. Small molecular fluorescent probes have been widely used for cell detec-tion, and fluorescence measurements, such as fluorescence intensity and lifetimes are two of the most promising methods for on-chip cell analysis [43,44]. However, the widely used small-molecule indicators typically possess problems, such as fast leakage, lack of membrane permeability, poor photostability, or sensitivity to ionic strength [45], and these problems have limited their practical applications. To over-come these problems, fluorescence-based microrobots have potential as sensors in medicine and biotechnology, especially since multiple indicators can be attached to a single particle. To measure extracellular and intracellular environment, our group has proposed fluorescent microrobots which are dyed with environment-sensitive indicator or fluorescence reagent [7,46]. However, the effect of other conditions on sensitivity to the target condition has not been sufficiently considered [47]. For example, many pH chemical sensors were investigated based on the fluorescence property of fluorescein isothiocyanate (FITC) [48], but the sensitivity of FITC to temperature was not adequately considered.

In this section, we introduce the sensing technique of cellular environment based on a fluorescent microrobot which is dyed with environment-sensitive fluorescent dye. This technology provides stable fluorescent measurement and the robots can be manipulated by using optical tweezers. Thus, extracellular or intracellular environ-ment can be measured at arbitrary positions with long time stability by using the proposed robots.

4.2 CONCEPT OF OPTICAL MULTISENSING MICROROBOTWe fabricated optical multisensing microrobots based on polymeric microbeads which can respond to both pH and temperature change of environments [10]. Two different kinds of fluorescent dyes (Rhodamine B and FITC) are introduced to a single microbead simultaneously, but the positions of FITC and Rhodamine B are different, as shown in Fig. 7.26. Central part of the robots is stained with Rhodamine

FIGURE 7.26 Schematic Illustration of the Synthetic Process for the Optical Multisensing Microrobot [10]

Copyright 2014 Elsevier. Reprinted with permission from Liu H. et al. Sensors Actuators B Chem 2014;203:

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222 CHAPTER 7 Optically driven micro- and nanorobots

B and edge part is stained with FITC. So interference from each fluorescent dye is expected to be negligible by this method.

4.3 MATERIALS AND METHOD4.3.1 Synthesis of the optical multisensing microrobotPolystyrene (Ps) microbeads (1 µm in diameter) with amino group modified surfaces were used as the sensor carriers. Rhodamine B was embedded inside the amino-polystyrene beads, and FITC was modified on the surface of the beads as shown in Fig. 7.26. First, a solution of amino-polystyrene beads and 1 g/L Rhodamine B (in alcohol) (1:1 v/v) was stirred for 5 min and then washed with deionized (DI) water. Then, the beads were added to a FITC saturated aqueous solution for 1 h and followed by three washes with DI water. FITC was immobilized on the surface of the amino-polystyrene beads through the chemical reaction shown in Fig. 7.27. The positions of FITC and Rhodamine B are different so interference from each fluorescent dye is expected to be negligible, which is confirmed by the experimental results shown in Fig. 7.28.

4.3.2 Experimental systemsThe fluorescent image of the target is obtained from an inverted confocal microscope (Ti-E Nikon) equipped with a high magnification lens (Plan Fluor 100 ×, Nikon) and CCD camera (iXon ultra, Andor). The excitation wavelengths of FITC are in the range of 405–525 nm with strong absorbance at 488 nm. The excitation wavelengths of Rho-damine B are in the range of 500–590 nm with strong absorbance at 561 nm. Therefore, wavelengths of 488 and 561 nm were selected as the excitation wavelengths for FITC and Rhodamine B, respectively. Intensity based fluorescence measurements depend on many parameters, and the fluorescence intensity is represented in Eq. (7.19) [49].

τ= ′ Φ ∈ − ∈ × −

I t I C xC

t( ) exp( ) exp0

(7.19)

where I (W/m3) is the optical energy emitted from the fluorescent material per unit time, ′Io (W/m3) the excitation light flux on the fluorescent material, C (g/m3) is the

I(t)=I′0CΦ∈exp(−∈xC)×exp−tτ

I′0

FIGURE 7.27 Process for FITC Assembly on the Amino-Polystyrene Beads [10]

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2234 Sensing technology for light robotics

concentration of the fluorescent material, Φ is the fluorescence quantum yield, and ∈ is the absorbance index. x(m) is the propagation distance in the material. τ(s) is fluorescence lifetime. t(s) is the excitation time. Φ is variable depending on envi-ronmental conditions, such as temperature, pH, and ions. The excitation lasers are controlled using a laser confocal scanning unit (CSU-X1, Yokogawa).

4.3.3 Measurement methods of the microrobotWe studied the fluorescence responses of the microrobot to different pH values and temperatures. Buffer solutions with different pH values (pH 5–8) were prepared. The temperature was controlled in the range of 32–38°C. The fluorescence intensity of the beads was measured using a fluorescence microscope. For the fluorescence measurements of the microrobot response to pH and temperature, the sample tem-perature was increased from 32 to 38°C in a pH 5 solution. Then the pH was changed to 6, 7, 8, and the fluorescence responses to temperature (32–38°C) were repeatedly measured. We kept detecting the same beads during the experiments.

4.4 EVALUATION RESULTS OF THE OPTICAL MULTISENSING MICROROBOT4.4.1 Response of Rhodamine B to pH and temperatureThe fluorescence responses of Rhodamine B to pH and temperature were measured in different pH solutions and at different temperatures. We defined the fluorescence

FIGURE 7.28

Fluorescent images for indicators (A) FITC (excited at 488 nm) and (B) Rhodamine B (excited at 561 nm) [10].Copyright 2014 Elsevier. Reprinted with permission from Liu H. et al. Sensors Actuators B Chem 2014;203:

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224 CHAPTER 7 Optically driven micro- and nanorobots

intensity detected in a pH 5 solution at 32°C as the basis point (F0), and the relative fluorescence intensity (F) is the normalized value by comparing the measured fluo-rescence intensity (F1) to the basis point (F0). The relative fluorescence intensity (F) is a nondimensional value since it is the ratio of F1 and F0. ∆F is the change in the value of F. As shown in Fig. 7.29, the relative fluorescence intensity of Rhodamine B decreased as the temperature increased, and the sensitivities obtained in the dif-ferent pH solutions were the same. It should be emphasized that Rhodamine B has a linear relationship between its relative fluorescence intensity and temperature, and it is independent of pH. Its relationship is shown in Eq. (7.20). The temperature infor-mation can be calibrated based on the fluorescence change in Rhodamine B using Eq. (7.20).

∆ = − × ∆F T0.034R(Rho.B) (7.20)

4.4.2 Responses of FITC to pH and temperatureAccording to the measurement procedure, the temperature of the chamber was increased from 32 to 38°C, and the fluorescence responses of FITC to tempera-ture were measured in solutions with different pH values (5–8). Several curves of the relative fluorescence intensity based on the temperature in solutions with different pH values were obtained and are shown in Fig. 7.30. It can be seen in Fig. 7.30A that the relative fluorescence intensity of FITC decreases as the tem-perature increases in the range from 32 to 38°C, and a linear relationship between the relative fluorescence intensity and temperature was found and is expressed as Eq. (7.21). After a linear fitting of Fig. 7.30A, the temperature sensitivities of FITC are shown in Fig. 7.30B. It should also be noted that the temperature sensitivities of

∆FR(Rho.B)=−0.034×∆T

FIGURE 7.29 The Response of Rhodamine B to Temperature at Different pH (5–8) [10]

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2254 Sensing technology for light robotics

FITC are dependent on the pH, and the temperature sensitivity f(pH) is expressed by Eq. (7.22). It should be emphasized that FITC is dependent on both temperature and pH. Based on Fig. 7.30, the fluorescence responses of FITC to pH at different temperatures can also be obtained and are shown in Fig. 7.31. It is obvious that the pH sensitivity of FITC is also dependent on temperature. The pH information can

FIGURE 7.30

(A) Responses of FITC to temperature at different pH values (5–8), (B) Temperature sensitivity of FITC at different pH values (5–8) [10].Copyright 2014 Elsevier. Reprinted with permission from Liu H. et al. Sensors Actuators B Chem 2014;203:

54–62.

FIGURE 7.31

(A) Responses of FITC to pH at different temperatures (32–38°C), (B) pH sensitivity of FITC at different temperatures (32–38°C) [10].Copyright 2014 Elsevier. Reprinted with permission from Liu H. et al. Sensors Actuators B Chem 2014;203:

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226 CHAPTER 7 Optically driven micro- and nanorobots

be calibrated based on the relative fluorescence change of FITC using Eq. (7.23), and the pH sensitivity, g(T), related to temperature is expressed as Eq. (7.24). These linear relationships are necessary for the temperature compensation required for pH calibrations.

∆ = × ∆F f pH T( )R(FIT C) (7.21)

= × −f pH pH( ) 0.0499 0.445 (7.22)

∆ = × ∆F g T pH( )R(FIT C) (7.23)

= × +g T Temp( ) 0.047 1.93 (7.24)

= ∆ + =∆

+pH pH pHF

g T( ) pH2 1R(FIT C)

1 (7.25)

where pH1 is the initial pH value, pH2 the pH value after change, and ∆pH is the change in the pH value.

4.4.3 pH and temperature measurements by the microrobot using temperature compensationIn order to confirm the accuracy of the microrobot for pH and temperature change calibrations, the fluorescence responses of the microrobot to changes in pH and temperature were measured. The pH values and temperature of the solution were ch a-nged as (1) pH 8, 32°C → (2) pH 7, 32°C → (3) pH 7, 34°C → (4) pH 6, 34°C → (5) pH 6, 36°C → (6) pH 5, 36°C → (7) pH 5, 38°C and then returned to (1) in opposite order. The fluorescence responses of Rhodamine B and FITC to pH and temperature changes are shown in Fig. 7.32. Fig. 7.32A shows that Rhodamine B only responds to temperature changes, which is consistent with the results in Fig. 7.29. Fig. 7.32B also shows that a decrease in pH or increase in temperature can induce a decrease in the fluorescence intensity of FITC. Moreover, it can also be seen in Fig. 7.32 that the fluorescence intensities of Rhodamine B and FITC return to their original values after the pH and temperature parameters return to the original condition.

Based on the measured relative fluorescence intensity changes in Fig. 7.32 and Eqs. (7.20) and (7.24), the temperature and pH values of the solutions can be cali-brated. As shown in Fig. 7.33, based on the fluorescence changes in Rhodamine B, the temperature was calibrated using Eq. (7.20). Fig. 7.33B shows that the calibrated temperature values are consistent with the measured values, and the accuracy of the temperature calibration by Rhodamine B is within 0.1°C. For FITC, which can respond to both pH and temperature changes, temperature compensation was neces-sary for pH calibration to remove any interference from its temperature response. Its fluorescence change with pH and temperature is shown by the black data in

∆FR(FIT C)=f(pH)×∆T

f(pH)=0.0499×pH−0.445

∆FR(FIT C)=g(T)×∆pH

g(T)=0.047×Temp+1.93

pH2=∆pH+pH1=∆FR(FIT C)g(T)+pH1

2274 Sensing technology for light robotics

Fig. 7.34A. The fluorescence changes of FITC caused by temperature changes can be calibrated using Eq. (7.21). Once the fluorescence change caused by the tempera-ture was added to the black data, the fluorescence change of FITC with tempera-ture compensation is shown as the red data. Based on the results plotted with black

FIGURE 7.32

Fluorescence responses of the microrobot to pH and temperature changes (A) Rhodamine B (B) FITC [10].Copyright 2014 Elsevier. Reprinted with permission from Liu H. et al. Sensors Actuators B Chem 2014;203:

54–62.

FIGURE 7.33

(A) Fluorescence responses of Rhodamine B to temperature and pH. (B) The measured and calibrated temperature values based on the fluorescence response results in (A) [10].Copyright 2014 Elsevier. Reprinted with permission from Liu H. et al. Sensors Actuators B Chem 2014;203:

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square, the pH values of the solution, with and without temperature compensation, were shown in Fig. 7.34B. After temperature compensation by our proposed method, the pH accuracy based on the calibrated fluorescence change of FITC was improved from 1.5 to 0.2.

4.5 SUMMARYWe fabricated optical multisensing microrobots based on polymeric microbeads that can simultaneously support pH and temperature sensitive FITC dye and tem-perature sensitive Rhodamine B dye on a single particle. Rhodamine B possesses high fluorescence emission at an excitation wavelength of 561 nm and shows good fluorescence responses to temperature in the range of 32–38°C. The calibrated sensitivity of Rhodamine B is −3.4%/°C, with a temperature accuracy of 0.1°C. FITC possesses high fluorescence emission at an excitation wavelength of 488 nm and shows good fluorescence responses to solution pH. Because temperature can also affect the fluorescence change of FITC, we proposed a method to perform a temperature compensation for pH calibration. After the temperature compensa-tion by our proposed method, the calibrated pH value based on the fluorescence change in FITC was consistent with the measured value. The pH accuracy was improved from 1.5 to 0.2. Using this microrobot, pH and temperature changes can be calibrated. This microrobot has high selectivity for pH and temperature, good stability, and a high tolerance for ionic strength, making it suitable for cellular measurements. The microrobot is based on a single microbead, which would only be a small stimulus to cells and could also detect the local cell conditions. The proposed microrobot not only can be used in cell measurement, but also sensing in

FIGURE 7.34

(A) Fluorescence responses of FITC to temperature and pH. (B) The measured and calibrated pH values based on the fluorescence response results in (A) [10].Copyright 2014 Elsevier. Reprinted with permission from Liu H. et al. Sensors Actuators B Chem 2014;203:

54–62.

2295 Biomedical applications of light robotics

other closed microenvironments, such as microchamber or microfluidic chip. It can provide low contamination and high accuracy for local condition measurement in microenvironments.

5 BIOMEDICAL APPLICATIONS OF LIGHT ROBOTICS5.1 INTRODUCTIONIn the previous sections, we explain technologies for manipulation, fabrication, and sensing of optically driven micro- and nanorobots. In this section, we introduce applications of these technologies, such as stiffness measurement of red blood cells (RBC) [9], single cell puncture [8], or pH measurement of virus infected cells [50].

5.2 MEASUREMENT OF CONTACT FORCE OF MICROROBOT TO RED BLOOD CELLSAs we explained in Section 2, we constructed multiple-trap manipulation and force feedback system of optical tweezers. For evaluating the effectiveness of the system for practical application, the demonstration of touching red blood cells of horses was performed. The output laser power from objective lens was set to 100 mW and the optical trap stiffness Kt was 64 pN/µm. Force feedback gain sf was set to 2.5 × 1010 for generating strong force feedback because the error between slave forces and actual forces was reduced by using modified slave forces. This teleoperation was done by squeezing red blood cells with two slaves as shown in Fig. 7.35. Here, two red blood cells which adhere to each other were caught by the slaves. The modified slave forces are indicated as force vectors at the center of slaves. Fig. 7.35 shows that the contact of slaves to the red blood cells was successfully detected.

5.3 SINGLE CELL PUNCTURE BY USING LASER HEATING OF CNTCell puncture or poration is one of the most important cell surgeries, which is used to inject DNA, siRNA, or fluorescent probes into a cell. Chapter 10 discusses various methods for cell poration. However, cell puncture for a single somatic cell has never been realized by previous on-chip robots because it requires high positioning accu-racy and large force. Therefore, we demonstrated such a single-cell puncture with the hybrid nanorobot which is explained in Section 3 by utilizing the high photothermal characteristics of the CNTs.

In this study, we used the Madin–Darby canine kidney (MDCK) cells as targets and stained them with a fluorescent dye (Life Technologies Corporation, Cell Mask Plasma Membrane Stain), which can selectively stain the cell membrane. The cell membrane staining was conducted to confirm the cell membrane shape and to recog-nize the inside and outside of the cell. Additionally, we introduced fluorescent nano-beads (Thermo Fisher Scientific Inc., G100, 100-nm diameter) into the solution in a microfluidic chip. It has been reported that if the target cell is punctured, a fluorescent

230 CHAPTER 7 Optically driven micro- and nanorobots

reagent in the culture medium can be introduced into it [41,42]. Therefore, we used fluorescent nanobeads to confirm if the target cell was successfully punctured by observing fluorescent nanobeads inside the cell. The details of the experimental pro-tocol are described as follows (Fig. 7.36):

1. The nanorobot is fixed on the glass substrate, and a target cell is transported to the CNT probe by using HOT.

2. When the target cell is attached to the CNT probe, the CNT is irradiated by the IR laser.

3. The dispersed nanobeads are introduced into the target cell through a punctured point.

4. The stained cell membrane and nanobeads are observed with a laser confocal fluorescent microscope.

Fig. 7.13 shows bright field (BF) images (A); the fluorescent image of a stained cell membrane (B) (excited by 561-nm laser); and a fluorescent image of the nano-beads [(C), excited by 488-nm laser] after the CNT was irradiated by the laser. The fluorescent signal from the nanobeads was detected inside the target cell, as indicated

FIGURE 7.35 Demonstration of Bilateral Teleoperation of Touching Red Blood Cells

Upper images show slave movements and lower graph shows the magnitude of modified slave forces [9].

Copyright 2012 Optical Society of America. Reprinted with permission from Onda K, Arai F. Opt Express

2012;20(4):3633–3641.

2315 Biomedical applications of light robotics

in Fig. 7.13C. Therefore, it is thought that the target cell was successfully punctured and that the nanobeads were introduced through the punctured point (Fig. 7.37).

5.4 MEASUREMENT OF PH ON VIRUS-INFECTED CELL MEMBRANEAs an application of optical multisensing microrobot for sensing of pH and tem-perature, we measured pH value of influenza virus infected cells [50]. The influenza virus can infect a wide range of vertebrate species. Several studies have reported that

FIGURE 7.36 Experimental Protocol of Cell Puncture [8]

Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

FIGURE 7.37 Result of Cell Puncture by the Hybrid Nanorobot

(A) Bright field. (B) Cell membrane (561 nm). (C) Nanobeads (488 nm)[8].Copyright 2014 IEEE. Reprinted with permission from Hayakawa et al. IEEE Trans Robot 2014;30(1):59–67.

232 CHAPTER 7 Optically driven micro- and nanorobots

large amounts of RNA are synthesized within a short period after the influenza virus enters the cell, thereby suggesting that the rate of ATP consumption would be higher in influenza virus-infected cells than in uninfected cells [51,52]. Some researchers have reported a reduction in intracellular pH of virus-infected cells [53,54]. But no researches have reported the extracellular pH change induced by virus infection. The investigation on pH can improve our understanding of the metabolic pathway of the cell and also the pH gradients inside and outside the cell membrane.

Fig. 7.38A,D show virus-bound and virus-unbound MDCK cells, respectively. The virus is stained by the fluorescence of Syto 21. After 15 min incubation of virus with MDCK cells, some of the viruses were bound to the cell membranes by using optical tweezers, following which the medium containing unbound virus was replaced with a new medium. The microrobot was then attached to virus-bound and virus-unbound cells using optical tweezers (Fig. 7.38B,E), and the fluorescence of Rhodamine B and FITC can be observed under excitation sources of 532 and 470 nm, respectively. Fig. 7.38C,F show the fluorescence of Rhodamine B of the microrobot, with no fluorescence observed from Syto 21 of the virus.

FIGURE 7.38 Fluorescence Images of the Microrobot and Virus on the Cell Membrane

(A) Virus adhered to the cell surface. (B) A microrobot adhered to the same cell. (C) A microrobot adhered to the same cell with a virus on its surface. (D) Virus-unbound cell. (E) A microrobot adhered to a virus-unbound cell. (F) A microrobot adhered to a virus-unbound cell [50].

Copyright 2016 Liu H, Maruyama H, Masuda T, Honda A, Arai F. This is an open-access article distributed

under the terms of the Creative Commons Attribution License (CC BY).

2335 Biomedical applications of light robotics

Based on the sensitivity of FITC to pH changes as evaluated in Section 4, pH changes can be calculated directly from the fluorescence intensity changes of FITC. Here, it is important to note that there will be changes in cellular temperature after infection with the virus. Several studies have reported that large amounts of RNA are synthesized within a short period after the influenza virus enters the cell. Thereby, suggesting that the rate of ATP consumption would be higher in influenza virus-infected cells than in uninfected cells [51,52]. A higher rate of ATP consumption compared to its metabolism in the cell will likely increase the temperature of the virus-bound cell. This temperature change can also be responsible for changing the FITC fluorescence. Therefore, temperature compensation is necessary for pH calcu-lation, as evaluated in Section 4.

Fig. 7.39A,B show pH changes of the virus-bound and unbound cells after tem-perature compensation. Fig. 7.39A shows the pH of virus-bound cell decreased from 2 h after the virus adhesion. The pH values of influenza virus-bound cell decreased by approximately 0.55 in 4 h after virus binding. However, there was no obvious change in the pH values of the virus-unbound cell as shown in Fig. 7.39B. These results suggest that the influenza virus infection and proliferation in the host cell could induce a pH decrease near the cell membrane.

5.5 SUMMARYIn this chapter, we explain manipulation, fabrication, and sensing technologies for optically driven micro- and nanorobots. Technologies of these optically driven micro- and nanorobots enable various cell manipulations, which is impossible with simple manipulation system and tools of optical tweezers. Furthermore, we intro-duce recent biological applications of these technologies, such as force measurement

FIGURE 7.39

The average pHe changes of (A) virus-bound cells and (B) virus-unbound cells (n = 8) [50].

Copyright 2016 Liu H, Maruyama H, Masuda T, Honda A, Arai F. This is an open-access article distributed

under the terms of the Creative Commons Attribution License (CC BY).

234 CHAPTER 7 Optically driven micro- and nanorobots

of RBC, single cell puncture or pH measurement of influenza virus infected cells. These results of applications indicate the strong potential that optically driven micro- and nanorobots will strongly contribute to micro- and nanobiotechnology fields in the future.

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237

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00008-2Copyright © 2017 Elsevier Ltd. All rights reserved.

Anton Sergeyev, Rachel GrangeOptical Nanomaterial Group, Institute for Quantum Electronics, Zurich, Switzerland

CHAPTER OUTLINE

1 Introduction ...................................................................................................... 2372 State of the Art .................................................................................................. 239

2.1 Optical Application of Nanowires .....................................................2392.2 Fabrication of LiNbO3 Nanowires .....................................................240

3 Theoretical Background .................................................................................... 2423.1 SHG and Phase-Matching ...............................................................242

4 Sample Fabrication ........................................................................................... 2445 Experimental Setup ........................................................................................... 2456 Experimental Results and Discussion ................................................................. 246

6.1 Demonstration of the SHG ..............................................................2466.2 Maximizing the Guided SHG Signal .................................................2476.3 Localized Dye Excitation .................................................................252

7 Conclusion and Outlook ..................................................................................... 256Acknowledgments .................................................................................................... 257References .............................................................................................................. 257

1 INTRODUCTIONThe capacity to optically trap and manipulate micro- and nanoscale constituents [1–3] is an essential element in the development of light robotics [4]. In analogy to the practice of attaching various tools and devices to robotic arms, one can use controlled laser beams to actuate and manipulate various microstructures. Besides mechanical manipulation, it is also possible to perform other tasks by trapping light-activated

Enhanced second-harmonic generation in lithium niobate nanowires used for localized light delivery

8

238 CHAPTER 8 Enhanced second-harmonic generation

micro- and nanotools that are designed to achieve new and enhanced functionalities. To do so, one has to merge various optical fields, such as nonlinear optics that is a rich resource for creating these new tools.

The field of nonlinear optics took off 1 year after the invention of lasers when second-harmonic generation (SHG) was demonstrated in quartz [5]. In the last 50 years, several applications of the SHG have emerged in our daily life to widen the laser sources [6–8] and perform SHG microscopy of living tissue [9–11]. However, nonlinear optical applications require high power consumption and high interaction length within bulk materials. As a result, either materials with stronger nonlinearity or strategies to enhance the electromagnetic field have to be found. Nanostructured materials may provide one of the potential solutions to enhance the electromagnetic field.

Thanks to their sizes, nanomaterials allow a stronger light-matter interaction. For example, Chapter 7 embedded carbon nanotubes (CNT) into designed microstruc-turess and utilized the CNT’s strong photothermal effect to indirectly puncture cells with laser illumination. Nanomaterials possess scattering resonances [12,13,13a], cavity resonances [14–16], and strong confinement of light [17,18], which enhance the efficiency of the light-matter interaction. Consequently, the SHG is already ex-tensively studied in nanomaterials of various shapes. For example, SHG is already demonstrated in single nanoparticles of such materials as BaTiO3, [13a,19–21] LiNbO3, [22] KNbO3 [23–25], BiFeO3, [26] ZnO, [21,27], and KTiOPO4 [28]. More-over, the SHG signal in nanoparticles can be even enhanced by combining dielectric nanoparticles with metal shells [29–32] and antennas [33].

SHG can be also locally enhanced in nanomaterials with cavities and varying cross-sections. Such nanostructures allow local confinement of light thanks to the Fabry-Perot [16] and whispering-gallery modes [14–16]. As a result, an enhanced SHG signal is obtained in a certain regions of the nanostructure and can be further used for characterization [34] or sensing [35–37] purposes.

There is also a large number of the SHG experiments on nanowires. Because of their strong nonlinear properties, [38] semiconductor nanowires are mostly studied for scattering and confinement of the SHG light [34,39–41]. However, most semi-conductor nanowires absorb light in the visible range. Therefore, there are only few works on waveguiding of the nonlinear signal in the semiconductor nanow-ires [42,43]. Thus, nanowires of other materials should be used for the purpose of generating and waveguiding the nonlinear signal. The LiNbO3 crystal is one of the potential materials since it is transparent in a broad wavelength range [44] and shows relatively strong nonlinearity [38].

In this chapter, we focus on LiNbO3 nanowires. First, we review the applications and fabrication methods of LiNbO3 nanowires. These applications provide a glimpse of functionalities, which can be potentially integrated into light robotics. Further, we show our study on SHG in LiNbO3 nanowires. Exploiting the SHG in these nanow-ires means that we can activate and control them using longer wavelengths, which can penetrate better into scattering materials like biological tissues. We demonstrate waveguiding and generation of the SHG and discuss the ways to enhance the SHG

2392 State of the art

signal. The higher energy light emitted from the nanowire can then be used to se-lectively excite targets in the host materials. Finally, we demonstrate localized ex-citation of fluorophore using the guided SHG light from a LiNbO3 nanowire. These properties of the nanowires can potentially play an important role in advancing light robotics applications for nanobiophotonics.

2 STATE OF THE ARTIn this chapter, we give an overview over the optical applications of nanowires and existing fabrication methods of LiNbO3 nanostructures.

2.1 OPTICAL APPLICATION OF NANOWIRESSubwavelength size of nanowires makes possible new effects in light-matter interac-tion, which result in new applications [45–47]. For example, efficient lasing is achieved in single semiconductor nanowires, which can be applied for lab-on-chip applications [48–52]. Single nanowires are also shown to directionally scatter excited photolumi-nescent light, which can be used for development of light sources [53–55]. Besides, knowing the scattering directionality allows to enhance efficiency of light absorption in nanowires and, thus, further develop solar cells [56] and sensing [57] applications.

Despite the submicron size of the nanowire cross-section, efficient coupling and waveguiding of light is also demonstrated [58]. The guiding ability gives rise to a number of effects and applications of nanowires. The guiding process in a single semiconductor nanowire can be controlled and switched by external light source [59]. Thus, nanowires can be used for developing optical logical elements [42,59]. Moreover, due to the nanoscale size of nanowires, large part of the guided modes is located outside the nanowire in terms of evanescent field [58]. Thus, nanowire can be used as optical sensors for the analysis of chemical substances [46,60] or light sources for localized imaging by exciting fluorescent material with evanescent field [17,61,62]. Since each guided mode has different fractions of evanescent field, the earlier-discussed sensing application can be further optimized by switching guided modes [58]. The guided modes can be switched either by varying the coupling condi-tions [58,63] or by structuring waveguides [64,65]. Furthermore, localized imaging applications can be also realized by the guided signal at the nanowire output [66,67]. Due to a high spreading angle, [3,51,58] the guided light excites the fluorescent material only in the vicinity of the output facet of the nanowire. The position of a nanowire can be manipulated by a laser beam through optical trapping to scan the sample [2,3,68,69]. Moreover, nanowire can be used not only for delivering light into a sample but also for collecting the signal from the sample and guiding it back for analysis [66].

For further development of the nanowire applications and complementation of the existing ones, nonlinear wave mixing effects, such as SHG can be applied [45,70]. For example, using the SHG process may expand the localized imaging

240 CHAPTER 8 Enhanced second-harmonic generation

applications [69,71]. In this case, the guided second-harmonic (SH) light is used for exciting the fluorescent material. Whereas the powerful laser beam, which is used for manipulating nanowire position, is spectrally shifted into the near-infrared (NIR) range. As a result, the fluorescent material will be neither excited, nor bleached by the laser beam but only by the SH light. Similarly, the SHG process can be used for nanowire microscopy applications [69]. In this application, the sample is scanned by an optically-trapped nanowire and by controlling the transmitted SH light, one can deduce information about the sample topography. Furthermore, based on nonlinear wave mixing, such as difference-frequency generation [42] and sum-frequency gen-eration [72], nanowires can be used as nonlinear light sources and be further applied for lab-on-a-chip applications.

Most of the earlier-discussed optical experiments are demonstrated in semicon-ductor nanowires, such as GaAs, [34,73], CdS [42], GaP [63,74,75], AlN [43,76], SnO2 [66], and ZnO [16,77]. However, the semiconductor material show absorption in the visible range, as a result, the application range of nanowires is limited. On the contrary, such materials as LiNbO3 possess a wide transparency in visible and infrared ranges (from 0.33 to 5.5 µm) [44]. Moreover, the LiNbO3 nanowires have been shown to efficiently generate [71,78] and guide the SH light [65,79,80]. Fur-thermore, LiNbO3 nanoparticles are proved to be nontoxic [81] and, thus, they can be used for biological applications.

2.2 FABRICATION OF LiNbO3 NANOWIRESThe LiNbO3 nanowires can be obtained through either bottom-up or top-down processes. Later, we give an overview over the possible synthesis and fabrication methods.

2.2.1 Bottom-up chemical synthesis methodsThere are mainly two bottom-up methods for synthesizing LiNbO3 nanowires, which are different from typical semiconductor nanowire synthesis methods [82–84]. The LiNbO3 nanowires can be fabricated either through the molten sol-gel method [79,85,86] or hydrothermal method [71,78,79].

In the molten sol-gel method, the oxides of the LiNbO3 constituents are used. The mixture of the oxides is mixed and heated up above the melting temperature. After the oxides are melted and further mixed, LiNbO3 starts nucleating and nanowires can grow. The molten sol-gel method is also used for synthesizing other perovskite nanowires, such as KNbO3 and NaNbO3 nanowires [79].

In the hydrothermal method, temperature and pressure are used to synthesize nanowires. For this goal, powders of lithium hydroxide and niobium pentoxide are mixed in distilled water. When heating up the mixture and applying pressure, LiNbO3 starts to crystallize and precipitate. This process allows to synthesize nanoparticles of various shapes including nanowires, nanoparticles, and nanocubes [79]. The ob-tained structures depend on the concentration, fill-factor, time, oven temperature, and applied pressure [71,79].

2412 State of the art

The molten sol-gel method and the hydrothermal methods have been used to syn-thesize LiNbO3 nanowires with an aspect ratio up to 10 and 14, respectively [78,79]. Furthermore, the synthesized LiNbO3 nanowires have been shown to generate [78] and guide the SH [79].

Nevertheless, the described synthesis methods do not give full control over such parameters of the nanowires as the cross-section size and the crystal structure, which are crucial for the nonlinear response of the nanowires.

2.2.2 Top-down fabrication methodsA wider range of top-down methods have been developed for fabricating LiNbO3 waveguides and nanowires. There are several fabrication methods that allow submil-limeter buried and ridge waveguides. Buried waveguides are obtained by titanium indiffusion [87], proton exchange [88,89] and laser writing [90,91]. Ridge wave-guides are obtained by fluorine-containing gases plasma etching [92] and mechanical milling [93,94]. To fabricate micro- and nanowaveguides, one can find in literature, such methods as focused ion beam (FIB) milling [95], inductive coupled plasma reactive ion etching (ICP-RIE) [96], and ion-beam enhanced etching (IBEE) [97,98].

The FIB milling is a maskless fabrication method [95]. Instead of masks, it only uses a focused beam of highly energetic ions, which removes the material off the substrate. This method offers high precision and has been used to fabricate nanow-ires with the width down to 700 nm [95]. However, this method has several limits. First, it produces nanowires with inclined walls [95]. Second, this method is rela-tively slow and, as a result, is used for fabricating only individual nanowires.

To fabricate multiple nanowires simultaneously, ICP-RIE [96] and IBEE [97,98] methods are preferred. Both methods use protective masks, which are fabricated on the surface of a LiNbO3 membrane and define widths and positions of the to-be-fabricated waveguides. In the ICP-RIE method (Fig. 8.1A), argon-ion plasma is used to etch the open areas. In the IBEE method (Fig. 8.1B), argon-ion beam irradiation is used to amorphize the open areas, which lose their chemical resistivity and are removed with wet etching in hydrofluoric acid. In both methods, the mask-protected areas of the LiNbO3 stay undamaged and form waveguides. The thickness of the membrane defines the height of the nanowires.

All the earlier discussed top-down fabrication methods use a thin film of a LiN-bO3 crystal on an insulator substrate because these methods damage the LiNbO3 only at surface. In addition, the IBEE method allows using pure LiNbO3 wafers for fabri-cation if it is complemented with helium-ion beam irradiation (Fig. 8.1C). Since the helium ions have lighter mass, they penetrate into the LiNbO3 wafer and damage it at a certain depth, thus forming a buried layer [99–102]. The penetration depth depends on the energy of the ions and defines the height of the waveguides [98].

The ICP-RIE method was used to fabricate waveguides with the cross-section down to 700 nm × 4 µm [103]. In turn, the IBEE method was used to fabricate nanowires with the cross-section down to 50 × 50 nm2 [98]. However, the thinnest nanowire that was fabricated by the IBEE method and optically tested had the cross-section of 265 × 287 nm2 [98].

242 CHAPTER 8 Enhanced second-harmonic generation

3 THEORETICAL BACKGROUND3.1 SHG AND PHASE-MATCHINGThe SHG is a second-order nonlinear optical effect. It takes place in crystalline ma-terials and converts two photons of the same wavelength into a single photon with a halved wavelength and doubled energy (Fig. 8.2A) [38]. In Fig. 8.2B, we show the energy diagram of the SHG. Since the SHG uses virtual energy levels, there is no loss of energy and the response is instantaneous. Usually, the pump light and the gen-erated light are referred as fundamental harmonic (FH) and second harmonic (SH).

Since the SHG is a second-order nonlinear optical effect, it depends quadrati-cally on the incident light. Besides, it also depends on the second-order susceptibility tensor χ(2), which characterizes the ability of the medium to perform the second-order nonlinear optical wavemixing.

FIGURE 8.2

(A) Second-harmonic generation. (B) The energy diagram of the second-harmonic generation.

FIGURE 8.1 Top-Down Fabrication Methods of LiNbO3 Waveguides

(A) Inductive coupled plasma reactive ion etching, (B) ion-beam enhanced etching method, (C) ion-beam enhanced etching method including helium-ion beam irradiation.

2433 Theoretical background

λ χ λ( )

∝ ⋅P E

2,(2) 2

(8.1)

where λ

P

2 is the polarization density of the SH, χ(2) is the second-order suscepti-

bility tensor, and

λ( )E is the incident electric field at the FH.Besides the intensity of the incident light, the crystal structure of the used mate-

rial also plays an important role in the SHG process. Thus, the SHG can be only ob-tained in materials with a break in symmetry of the crystal structure [38]. As a result, the SH is generated either in bulk materials without crystal symmetry [38] or on the surface [63,74,75,104]. Moreover, since the components of the second-order suscep-tibility tensor differ from each other, one has to design the crystal structure and rotate the polarization of the incident pump light to assure that the largest components of the second-order susceptibility tensor are used.

In order to obtain a strong SHG in bulk materials, one has to take into account material dispersion. Because of the material dispersion, the refractive indices of the LiNbO3 vary for the FH and the SH. As a result, the FH and SH waves propagate with different phase-velocities and the SH waves that are generated at different positions along the propagation direction in the crystal may interact destructively. As a consequence, the overall SH may be weak at the output of the crystalline mate-rial. This effect is called phase-mismatching and is characterized by a wave-vector mismatch:

πλ

( )∆ = ⋅ − = − ⋅k k k n n24

,FH SH FH SHFH

(8.2)

where kFH—wavevector of the FH, kSH—wavevector of the SH, nFH—refractive index for the FH, nSH—refractive index for the SH, and λFH—wavelength of the FH.

In Fig. 8.3A, we show the behavior of the SH power in the cases of phase-mismatching (∆k≠0, dashed line) and phase-matching (∆k = 0, solid line). Plot-ted on different scales, the SH power in the case of phase-mismatching oscillates periodically along the propagation direction and never exceeds the case of phase-matching where the SH power constantly grows [38]. In Fig. 8.3B, we plot the SH intensity versus the pump wavelength around the phase-matching wavelength. The behavior follows the shape of a squared sinc-function with the center at the phase-matching wavelength.

To achieve phase-matching, there are several techniques developed. For example, one can use angle tuning [105], temperature heating [90], and periodical poling of the nonlinear crystal [106–108]. For waveguides, the geometry may allow modal phase-matching [109–111]. Modal phase-matching is based on the facts that light is guided in terms of modes in waveguides and each guided mode has a distinctive effective refractive index [112]. As a result, a pair of modes of the FH and SH may have the same refractive index at a certain pump wavelength and, consequently, the phase-matching.

P→λ2∝χ(2)⋅E→2λ,

P→λ2

E→λ

∆k=2⋅kFH−kSH=nFH−nSH⋅4πλFH,

244 CHAPTER 8 Enhanced second-harmonic generation

In waveguides, the efficiency of the SHG process depends on the wave-vector mismatch and conversion efficiency of the interacting FH and SH modes [113]. The conversion efficiency depends on the overlap integral of the FH and SH modes. Thus, for an efficient SHG process, one should phase-match the pairs of the FH and SH modes that have the largest overlap integral. Moreover, the generated SH field depends on the intensity of the FH field. In turn, the FH field depends on the pulse duration and on the coupling of the FH into a waveguide.

The nanowires, which we use for our experiments, have cross-sections around 500 × 500 nm2. Since the single-mode regime in LiNbO3 nanowires at the wave-length of 800 nm is expected to start only in cross-sections below 200 × 200 nm2, LiNbO3 nanowires provide a multimode regime in the spectral range of our experi-ments. As a result, several phase-matching effects with different modes are expected to be observed in our nanowires.

4 SAMPLE FABRICATIONIn the experiments discussed in this chapter, we use LiNbO3 nanowires that are fab-ricated through the IBEE [98]. The IBEE method was previously described in the state-of-the-art.

To prepare the mask, layers of silica, chromium, and e-beam resist are deposited onto an x-cut LiNbO3 wafer. The mask is first written into the e-beam resist with electron-beam lithography. Afterward, the mask is transferred into the layers of silica and chromium by reactive-ion etching.

After the mask is prepared, the wafer is irradiated with a series of argon ion beam, which amorphizes the uncovered regions of the wafer. The fluence and energy define

FIGURE 8.3

(A) Behavior of the SH along the propagation distance in the cases of phase-matching (solid line) and phase-mismatching (dashed line). (B) The SH power versus the pump wavelength. The behavior follows the shape of a sinc-function with the center at the phase-matching wavelength (λ = 846 nm in this case).

2455 Experimental setup

how deep the ion irradiation damages the crystal. After the argon ion irradiation, the mask is removed and the wafer is irradiated with helium ions. Since the helium ions are lighter, they penetrate into the wafer and create a buried layer of an amorphized LiNbO3 crystal. The penetration depth depends on the energy and fluence. The argon ions irradiation is performed at 600 keV energy with a fluence of 7.2 × 1014 cm−2 and 350, 150, 60 keV energies with a fluence of 1.38 × 1014 cm−2. The helium ion irradia-tion is performed at energy of 285 keV and fluence of 5 × 1016 cm−2.

After the irradiation, the amorphized regions are removed with wet etching in hydrofluoric acid, whereas the undamaged regions stay untouched and, thus the nanowires are formed. The fabricated nanowires are suspended in air and are fixed to the wafer only at their ends. We remove the nanowires off the wafer by sonicat-ing the wafer in ethanol. Thus, we obtain a solution of nanowires and ethanol. Later, we deposit the nanowires onto the glass by dropcasting several droplets onto a glass substrate. To enable the optical experiments and the scanning electron microscopy (SEM) measurement, we use prepatterned glass substrates, which are coated with an indium-tin-oxide film.

5 EXPERIMENTAL SETUPFor the optical characterization of the LiNbO3 nanowires, a home-made transmis-sion microscope is used. The schematic of the experimental setup is demonstrated in Fig. 8.4A. For the pump, we use a pulsed laser beam from a Ti:Sapphire oscillator (the pulse duration is around 295 fs at the setup entrance, the pulse repetition rate is 80 MHz and the tunable wavelength range is 690–1040 nm). Initially, the laser beam passes through a half-wave plate, which rotates the laser polarization. Afterward, a 10x objective (the focal distance is 10.6 mm and the numerical aperture is 0.25) focuses the laser beam onto the sample surface down to a beamspot with the FWHM of1.2 µm. We use a 100× objective (the working distance is 4 mm and the numerical aperture is 0.75) to collect the sample response and image it on an electron-multi-plying charge-coupled device (EMCCD) camera. At the EMCCD camera, we use bandpass filters to detect only the signal of interest.

In Fig. 8.4B, we illustrate a schematic of the laser coupling into a nanowire and the collection of the guided signal. The sample surface with the nanowires is located

FIGURE 8.4

(A) Schematic of the experimental setup for the SHG measurements. (B) The schematic of the laser coupling laser into a nanowire and the collection of the guided signal.

246 CHAPTER 8 Enhanced second-harmonic generation

perpendicularly to the optical axes of the focusing and collecting objectives. Thus, the laser is focused perpendicularly to the nanowire and only a fraction of the laser power is coupled into the nanowire. Similarly, we detect only a fraction of the guided signal that is scattered toward the collection objective [113a].

6 EXPERIMENTAL RESULTS AND DISCUSSION6.1 DEMONSTRATION OF THE SHGIn Fig. 8.5A, we show a white light picture of one of the studied LiNbO3 nanowires. According to the SEM measurements, the nanowire has the width of 460 ± 20 nm, the height of 610 ± 20 nm, and the length of 48.9 ± 0.4 µm. The x-, y- and z-axes of the nanowire are parallel to height, length, and width of the nanowire, respectively.

In the SHG experiments, we focus the laser beam on one of the nanowire ends. The laser beam has the power of 20 mW and the central wavelength of 790 nm. The beamspot is indicated with a dashed circle in Fig. 8.5A.

In Fig. 8.5B, we show the obtained SHG response from the nanowire, which disappears when the nanowire is removed out of the laser beamspot. Since the SHG signal is mostly observed at the input and the output of the nanowire, it indi-cates the waveguiding process. There is also a weak SH signal observed along the nanowire, which we refer to the scattering of the guided SH at roughness of the nanowire surfaces.

To prove the observed guided signal is, indeed, the generated SH, we measure the guided SH power at varied laser power and confirm that the guided signal quadrati-cally depends on the incident laser power (Fig. 8.5C). To estimate the power of the SH signal that is detected by the EMCCD camera, we first sum the signal of each pixel in the taken image and then back-convert the summation value by taking into account the quantum efficiency of the camera, acquisition parameters, and transmis-sivity of the experimental setup.

FIGURE 8.5

(A) The white light picture of the studied nanowire; (B) the SH signal from the nanowire. (C) Guided SH power versus the incident laser beam power.

Adapted from Sergeyev A, Geiss R, Solntsev AS, Steinbrück A, Schrempel F, Kley E-B, et al.

Second-harmonic generation in lithium niobate nanowires for local fluorescence excitation. Opt Express

2013;21:19012 [80] with permission from Optical Society of America.

2476 Experimental results and discussion

The coupling efficiency of the laser light into nanowires is low since the beamspot is significantly larger than the nanowire cross-section. We also expect that the cou-pling efficiency is influenced by the input facet of the nanowire and the polarization of the incident laser beam and also varies for each nanowire.

Besides the SH signals at the input and output of the nanowires, the camera also detects a weak SH signal along the nanowire. We attribute the observed signal to the scattering of the guided light by the roughness and imperfections of the nanowire surface [43]. As a result, the roughness of the nanowire causes losses for both FH and SH. We have not estimated the losses of the nanowires that are demonstrated in the chapter. Nevertheless, the losses of the guided FH light in similar waveguides are estimated to be 61 dB/cm [97].

6.2 MAXIMIZING THE GUIDED SHG SIGNALThe SHG efficiency quadratically depends on the volume of the nonlinear mate-rial [23,38]. As a result, the nanoscale structures provide a weak SH response and ways of enhancing the guided SH signal have to be found. Based on the theoretical background, we propose two different approaches for maximizing the guided SHG signal in the LiNbO3 nanowires [114]. In the first approach, we enhance the guided SH signal by using modal phase-matching. In the second approach, we optimize the guided SH signal by adjusting the nanowire length.

6.2.1 Modal phase-matching in LiNbO3 nanowiresTo test the phase-matching effect in nanowires, we measure the power of the guided SH signal versus the laser wavelength. In the measurement, the laser wavelength is varied in the range of 780–920 nm with the step of 5 nm, the incident power is kept at 3.4 mW and the polarization is kept at 20 degree with respect to the nanowire length. In Fig. 8.6, we show an SEM picture of the studied nanowire. According to the SEM measurement, the studied LiNbO3 nanowire has the width of 778 ± 38 nm, the height

FIGURE 8.6 SEM Image of the Studied Nanowire

Inset: Crystal structure of the nanowire.Reprinted with permission from Sergeyev A, Geiss R, Solntsev AS, Sukhorukov AA, Schrempel F,

Pertsch T, et al. Enhancing guided second-harmonic light in lithium niobate nanowires.

ACS Photonics 2015;2:687–691 [114]. Copyright 2015 American Chemical Society.

248 CHAPTER 8 Enhanced second-harmonic generation

of 541 ± 27 nm, and the length of 52 ± 2 µm. The x-, y-, and z-axes of the nanowire’s crystal structure are parallel to the height, the length, and the width, respectively.

The result of the measurement is plotted in Fig. 8.7 (dashed line with red squares). As one can see, the SH power strongly depends on the incident laser wavelength. Thus, Fig. 8.7 shows peaks at the pump laser wavelengths of 800 nm, 855 nm, and 900 nm. Thanks to these peaks, the SH can be significantly enhanced. For example, in the observed experiment, the guided SH is increased by a factor of 48, when the laser wavelength is tuned from 830 to 855 nm.

We refer the observed peaks to the phase-matching of three different pairs of the FH and SH modes. To confirm theoretically the phase-matching process in the studied nanowire, we simulate waveguiding and generation of the SH in a nanowire [113,114]. In the simulation, we calculate first 3 FH and 30 SH guided modes (with the use of a finite element method) and find conversion efficiencies for all possible pairs of the FH and SH modes. To simulate the total nanowire response of the guided SH, we sum up conversion efficiencies of all possible pairs of the FH and SH modes. For the calculation of the guided modes, we use wavelength-dependent Sellmeier equations for the ordinary and extraordinary refractive indices at the temperature of 23°C [115] and the second-order susceptibility tensor [38] of the LiNbO3 crystal.

The simulation is performed for all cross-sections within the uncertainty range of the SEM measurement. The best match between the simulation and the experiment curves is obtained at the width of 778 nm and the height of 560 nm. In Fig. 8.7 (solid line), we plot the calculated curve of the guided SH power versus the laser wave-length. Since the fractions of the laser power that are coupled into each FH mode are unknown, contributions of each FH mode are adjusted to fit the heights of the peaks in the theoretical and experimental curves. Nevertheless, there is still a slight lateral

FIGURE 8.7 Experiment and Simulation Results of the Guided SH Power Versus Incident Laser Wavelength

Reprinted with permission from Sergeyev A, Geiss R, Solntsev AS, Sukhorukov AA, Schrempel F, Pertsch T,

et al. Enhancing guided second-harmonic light in lithium niobate nanowires. ACS Photonics 2015;2:687–

691 [114]. Copyright 2015 American Chemical Society.

2496 Experimental results and discussion

discrepancy between the experiment and the simulation curves. This discrepancy can be explained by a high order of the guided modes, which are sensitive to slight varia-tions of the nanowire cross-sections [51,63]. Indeed, according to the simulations, the peak at 800 nm is created by the 1st FH mode and 17th SH mode, the peak at 855 is created by the 3rd FH mode and the 23rd SH mode and the peak at 900 nm is created by the 2nd FH mode and 17th SH modes. The order of the modes is sorted by the refractive index. The intensity profiles of the modes are shown in Fig. 8.8.

In conclusion, experimental and theoretical results confirm that the guided SH light can be phase-matched and, thus, get enhanced. Because of the phase-matching of multiple number of the FH and SH mode pairs, a strong SHG signal can be ob-tained in a broad wavelength range. The nanowire cross-section influences strongly both the position and the height of a phase-matching peak. Thus, the position and strength of the phase-matching peak can be engineered by careful design of the nanowire cross-section.

6.2.2 Adjustment of the nanowire lengthAs shown in Fig. 8.7, nanowires provide phase-matching possibilities only at a limited number of wavelengths. Thus, the SH signal may not be enhanced by the phase-matching effect at a desired wavelength in a particular nanowire. Nevertheless, nanowires may still provide pairs of the FH and SH modes that have a high conver-sion efficiency due to a large overlap integral. As a result, the power of the guid-ed phase-mismatched SH can be also optimized by adjusting the nanowire length, since it oscillates along the propagation distance. To demonstrate it, we calculate the

FIGURE 8.8

Intensity profiles of the 1st mode at 802 nm (A), 3rd mode at 846 nm (B), 2nd mode at 900 nm, the 17th mode at 401 nm (D), 23rd mode at 423 nm (E) and 17th mode at 450 nm (F).

250 CHAPTER 8 Enhanced second-harmonic generation

behavior of the guided SH power generated by the 1st FH mode at the pump wave-length of 820 nm in a nanowire with the height of 510 nm and the width of 728 nm. In the calculation we take into account the first 30 guided SH modes. We show the result of the calculation in Fig. 8.9.

The calculated result in Fig. 8.9, indeed, indicates a variation of the guided SH, which oscillates along the nanowire length. Moreover, after analyzing the obtained curve in Fig. 8.9, we conclude that the guided SH power mostly propagates in terms of three guided modes, which are the 4th, the 16th, and the 17th SH modes. These modes have the highest conversion efficiency with the 1st FH mode and, thus, give the highest contribution to the calculated guided SH power, which is shown in Fig. 8.9. The intensity profiles of the modes are shown in Fig. 8.10. We also plot the interaction behavior of the discussed SH modes with the 1st FH mode in Fig. 8.11.

Since the discussed SH modes are not phase-matched with the 1st FH mode, the conversion efficiency curves show oscillations. The periods correspond to the doubled value of the coherence lengths. Thus, the coherence lengths for the interac-tion of the 1st FH mode with the 4th, 16th, and 17th FH modes are 1 µm, 7.5 µm, and 6.5 µm, respectively. Since the coherence length depends on the refractive index mismatch of the interacting FH and (nFH−nSH) SH modes, it increases in the vicinity of the phase-matching regime.

Due to the multiple number of the guided SH modes, the behavior of the guided SH power gets more complicated and shows local minima and maxima. Consequent-ly, in this particular case, the power of the guided SH signal may vary by a factor of up to 21.2and the length of the nanowires has to be carefully adjusted to maximize the guided SH power.

FIGURE 8.9 Calculated Guided SH Power Generated by 1st FH Versus the Nanowire Length at the Pump Wavelength of 820 nm in a Nanowire With the Height of 510 nm and the Width of 728 nm

Adapted with permission from Sergeyev A, Geiss R, Solntsev AS, Sukhorukov AA, Schrempel F, Pertsch T,

et al. Enhancing guided second-harmonic light in lithium niobate nanowires. ACS Photonics 2015;2:687–

691 [114]. Copyright 2015 American Chemical Society.

2516 Experimental results and discussion

FIGURE 8.10

Intensity profiles of the 1st mode at 820 nm (A), 4th mode at 410 nm (B), 16th mode at 410 nm, and 17th mode at 410 nm (D).

FIGURE 8.11 Calculated Conversion Efficiency of the 1st FH Mode with the 4th, 16th, and 17th SH Modes

252 CHAPTER 8 Enhanced second-harmonic generation

To demonstrate it experimentally, we perform a series of experiments using a nanowire with the height of 728 ± 36 nm, width of 510 ± 26 nm, and initial length of 34.8 µm. In each experiment, we stepwisely shorten the nanowire with a FIB milling and measure the guided SH power after each milling procedure. The pump laser has the wavelength of 820 nm, power of 10 mW and polarization of 154° with respect to the nanowire length. Since the period of the phase-mismatched SH oscillation is un-known for this nanowire, the cutting steps are chosen arbitrarily and vary from 0.61 and 6 µm in this experiment. In Fig. 8.12, we plot the obtained guided SH power at various length of the nanowire. The result demonstrates that the guided SH power strongly depends on the nanowire length. Moreover, by cutting the nanowire, the guided SH power is increased by a factor of 9.3. Thus, one can optimize the phase-mismatched guided SH power by adjusting the nanowire length [114].

However, in order to know at which nanowire length the highest possible en-hancement can be obtained, one has to define the positions along the nanowire in which the guided SH power is maximal. This information can be obtained by mea-suring behavior of the near-field of the guided modes along the nanowire with a scanning near-field optical microscope [39,116–118].

6.3 LOCALIZED DYE EXCITATIONAs discussed earlier, nanowires can be used for a large application range includ-ing localized imaging by the guided SH. To demonstrate that the studied LiNbO3 nanowires can be used for localized imaging, we use 4’,6-diamidino-2-phenylindole (DAPI) dye, which is widely used in biological applications for staining the nuclei of cells [119,120]. The DAPI dye absorbs light below 500 nm with the absorption

FIGURE 8.12 Guided SH Power Versus the Nanowire Length

The dashed line is added to guide the eye.Reprinted with permission from Ref. Sergeyev A, Geiss R, Solntsev AS, Sukhorukov AA, Schrempel F, Pertsch

T, et al. Enhancing guided second-harmonic light in lithium niobate nanowires. ACS Photonics 2015;2:687–

691 [114]. Copyright 2015 American Chemical Society.

2536 Experimental results and discussion

peak at 340 nm [121]. As a result, we can use the NIR laser to generate SHG for the fluorescence excitation. Moreover, since the emission spectrum has maximum at 488 nm, [121] we easily differentiate the SH and the fluorescence signal by using appro-priate bandpass filters.

We prepare the dye solution by mixing water and DAPI dye in different con-centrations. In Fig. 8.13, we show the procedure of the sample preparation. We place nanowires on a glass slide (Fig. 8.13A) and add 10 µL of the dye solution (Fig. 8.13B). To prevent evaporation of the solution, we seal the sample with a cover slip (Fig. 8.5C). In all experiments, only freshly prepared samples are used, in order to prevent agglomeration of the dye.

For the dye excitation experiment, we use a laser beam at the wavelength of 760 nm and with the power of 85 mW to generate and guide the SH light at 380 nm. In Fig. 8.14, we show the obtained fluorescent signal excited by two nanowires, which are embedded in solution with dye concentrations of 1 µg/mL (A) and 50 ng/mL (B).

FIGURE 8.13 Procedure of the Dye Sample Preparation

(A) Placing nanowires onto the glass substrate. (B) Adding 10 µL of the dye solution. (C) Sealing the dye solution with a cover slip.

FIGURE 8.14

Images of the fluorescence signal in DAPI dye solution with concentrations of 1 µg/mL (A) and 50 ng/mL (B). The dashed and the dotted circles indicate the illumination area of the incident beam spot and the fluorescence signal at the nanowire output, respectively. The dash-dotted line indicates the position of the nanowire.

Adapted from Sergeyev A, Geiss R, Solntsev AS, Steinbrück A, Schrempel F, Kley E-B, et al. Second-

harmonic generation in lithium niobate nanowires for local fluorescence excitation. Opt Express

2013;21:19012 [80] with permission from Optical Society of America.

254 CHAPTER 8 Enhanced second-harmonic generation

In Fig. 8.14A,B, the fluorescence signal is excited at the input and at the output of the nanowires. At the input, the fluorescence signal is most probably excited by both the incoming laser beam through two-photon absorption (TPA) [122] and by the generated SH signal through single-photon absorption (SPA). At the output, the fluorescence signal is excited by a guided signal. We find important to note that the typical concentration for biological applications varies in a range from 50 ng/mL to 1.5 µg/mL [123,124]. Thus, LiNbO3 nanowires can be used for biological applications.

To differentiate if the fluorescence signal at the output is excited by the guided SH through SPA or the guided laser light through TPA, we measure the power of the guided laser light and SH for one of the tested nanowires. The guided laser light and SH power values are found to be 441 ± 44 nW and 2.57 ± 0.26 nW, respectively. Further, we take into account quantum efficiency [125], SPA and two-photon emis-sion cross-sections [126,127] of the DAPI dye and calculate that the fluorescence power that is excited by 441 ± 44 nW of the pump laser power and 2.57 nW of the SH power. According to the calculation, 441 nW of the laser power does not excite any fluorescence, whereas 2.57 nW of the guided SH should excite 1.028 ± 0.104 pW [80]. Thus, we conclude the fluorescence of the nanowire is excited by the guided SH signal.

For the localized imaging application, the imaging resolution is expected to de-pend on the nanowire cross-section. However, the SHG process quadratically de-pends on the volume of the crystalline material [23,38]. As a result, the generated SH signal gets weaker in thinner nanowires. The generated SH signal can be always increased by using a stronger pump power. However, the pump power is limited by the damage threshold power of the sample. Thus, the used nanowire should not have a cross-section below a certain limit for successful performance of the localized im-aging. Here, we theoretically estimate the smallest cross-section of a nanowire that can be used for efficient dye excitation in biological samples.

For this purpose, we calculate the maximum guided SH power that is generated with the laser beam in a nanowire with varied cross-sections. For the calculation, we first theoretically estimate the threshold power of a near-infrared laser beam for damaging a cell and experimentally estimate the smallest guided SH power for ef-ficient dye excitation.

To set the threshold power of a near-infrared laser light, we assume that the full laser power is coupled into a nanowire and limit the power of the guided laser beam with the damage threshold power of a cell. The damage threshold average power is estimated to be around 18.5 mW at the wavelength of 760 nm [128]. For the estima-tion, we assume a nanowire cross-section of 500 × 500 nm2 and pulse duration of 200 fs.

To estimate the smallest guided SH power for efficient fluorescence excitation, we perform two measurements with the same nanowire. In the first measurement, the studied nanowire is embedded in pure water and we quantify power of the guided FH and SH. In the second measurement, the nanowire is embedded in a dye solution with

2556 Experimental results and discussion

the DAPI concentration of 1 µg/mL and we quantify the power of the excited fluores-cent and find the signal-to-noise ratio (SNR) for the excited fluorescence signal. In Fig. 8.15A, we plot the calculate fluorescence power and the guided SH power versus the incident beam power. In Fig. 8.15B, we plot the SNR of the excited fluorescence signal versus the guided SH power. To define the SHG threshold power, we use the Rose criterion [129] and set the threshold of the SNR to 5. Thus, the threshold SHG power is estimated to be 63 ± 6 pW.

Having estimated the damage threshold power of the NIR laser and the threshold SH power for efficient dye excitation, we simulate generation and waveguiding of the SH signal. We perform calculation for the waveguides with the width and height of various cross-section size and crystal structure. We use the estimated laser power to find the maximum excited SH signal and compare with the estimated SH threshold power.

We find that nanowires with three crystal structures are able to generate the set threshold SH power below the cross-section 100 × 100 nm2. In Table 8.1, we list the smallest cross-section and crystal structures of the nanowires that are expected to provide the SH power above the set threshold power.

According to the results in Table 8.1, the smallest cross-section at which efficient dye excitation is possible is 40 × 60 nm2. Moreover, the existing fabrication meth-ods allow to fabricate nanowires down to 50 × 50 nm2 as we recently demonstrated [98]. As a result, the LiNbO3 have a potential for being used for localized imaging applications.

FIGURE 8.15

(A) Guided SH and excited fluorescence signal versus the incident laser beam power. The diamonds correspond to the guided SH and the circles correspond to the fluorescence signal. (B) SNR of the fluorescence signal versus the average power of the guided SH. The dashed line shows the limit value of the SNR equal to 5 at which the features of the image can be unambiguously identified according to the Rose criterion [129].

Adapted from Sergeyev A, Geiss R, Solntsev AS, Steinbrück A, Schrempel F, Kley E-B, et al. Second-

harmonic generation in lithium niobate nanowires for local fluorescence excitation. Opt Express

2013;21:19012 [80] with permission from Optical Society of America.

256 CHAPTER 8 Enhanced second-harmonic generation

7 CONCLUSION AND OUTLOOKWe have shown several aspects of our research on SHG in LiNbO3 nanowires. First, we have demonstrated generation and waveguiding of the SH in LiNbO3 nanowires. We have also discussed the ways of increasing the guided SH power by modal phase-matching or by adjusting the nanowire length. By modal phase-matching, the guided SH signal has been enhanced by a factor of 48. By adjusting the nanowire length, the guided SH has been increased by a factor of 9.3. Afterward, we have demonstrated fluorescent dye excitation by the guided SH in dye solution with concentration of 1 µg/mL and 50 ng/mL. Finally, we have theoretically estimated that nanowires with the cross-section down to 40 × 60 nm2 can provide enough SHG for efficient dye excitation without damaging biological samples.

As an outlook, we anticipate several further studies on nonlinear wave mixing in nanowires. First of all, ways to increase the coupling efficiency should be developed. We believe it can be achieved by shaping input facets of nanowires [42] and modify-ing the properties of the incident laser beam [63,65]. Second, the distribution of the guided light should be studied since this knowledge is of great importance for the imaging applications [69] and may help to increase their resolution. Moreover, we expect that one can engineer distribution of the guided light by shaping output facets of the nanowires [3]. Finally, nanowires can be further developed as nonlinear light sources based on nonlinear wave mixing. For example, sum-frequency-generation and difference-frequency generation effects may help to widen the spectral range of the generated light [129a]. As a result, nanowires can be used as nonlinear light sources, [42] optically switched logical gates [59] and measuring pulse duration [72].

Nanowires can interface with light robotics either via direct optical trapping and micromanipulation [2,3,69] or by embedding them into light-driven microrobots for improved control. Chapter 7 illustrates a technique for embedding nanotubes onto microrobots and subsequently using the photothermal effect in the nanotubes

Table 8.1 The Smallest Nanowire Height and Width for Various Nanowire Crystal Structures at Which the Nanowires Provides a Propagated SH Average Power Above 63 ± 6 pW and the Predicted Average Power of the SH Signal

The Crystal Structure Axes Parallel to the Nanowire Height and Width Sides

Height and Width of the Smallest Nanowire Cross-Section (nm2)

The Propagated SH Average Power Provided by the Corresponding Cross-Section (pW)

XZ 40 × 60 106.2XY 40 × 60 96.4ZY 60 × 60 113.9

Adapted from Sergeyev A, Geiss R, Solntsev AS, Steinbrück A, Schrempel F, Kley E-B, et al. Second-harmonic generation in lithium niobate nanowires for local fluorescence excitation. Opt Express 2013;21:19012 [80] with permission from Optical Society of America.

257

to puncture holes into cells. The optimized nanowires discussed in this chapter add new possibilities in light robotics and their integration with the various light-driven microrobots discussed in this book can open avenues for realizing new functional-ities in light robotics.

ACKNOWLEDGMENTSThe authors thank Marc Reig, Dr. Alexander S. Solntsev, Dr. Dragomir Neshev, and Dr. An-drea Steinbrück for their support and fruitful discussions. The authors also thank Reinhard Geiss and Prof. Dr. Thomas Pertsch for providing LiNbO3 nanowires. The authors acknowl-edge financial help from the Swiss National Science Foundation (grant 150609).

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265

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00009-4Copyright © 2017 Elsevier Ltd. All rights reserved.

Mark Jayson Villangca, Darwin Palima, Andrew Rafael Bañas, Jesper GlückstadTechnical University of Denmark, Lyngby, Denmark

CHAPTER OUTLINE

1 Introduction ...................................................................................................... 2652 Scaling of Physical Effects ................................................................................ 2663 Light-Driven Microrobots ................................................................................... 268

3.1 Harnessing Ideas From Optical Trapping and Micromanipulation ........2683.2 Turning Microstructures into Functional Microrobots ..........................270

4 Microrobots With Various Functional Loads ........................................................ 2724.1 Metallic Structures in Microfluidics .................................................2724.2 Hollow Microrobots for Material Transport .........................................2734.3 Microrobots for Chemical Sensing: Probe for Surface-Enhanced

Raman Spectroscopy ......................................................................2774.4 Microrobots for Temperature Sensing ...............................................279

5 Conclusion and Outlook ..................................................................................... 280References .............................................................................................................. 281

1 INTRODUCTIONConventional robotics provides machines and robots that can replace and surpass human performance in repetitive, difficult, and even dangerous tasks at industrial assembly lines, hazardous environments, or even at remote planets. A new class of robotic systems no longer aims to replace humans with so-called automatons but, rather, to create robots that can work alongside human operators. These new robots are intended to collaborate with humans—extending their abilities—from assisting workers on the factory floor to rehabilitating patients in their homes. In medical ro-botics, robot-assisted surgery imbibes surgeons with superhuman abilities and gives the expression “surgical precision” a whole new meaning. Still in its infancy, much remains to be done to improve human-robot collaboration both in realizing robots that can operate safely with humans and in training personnel that can work profi-ciently with the collaborative robots.

Next generation light robotics 9

266 CHAPTER 9 Next generation light robotics

Tracing its roots from optical micromanipulation, Light Robotics draws upon the strengths of these two enabling technologies—optics and robotics—to create prom-ising new tools to extend our control over, for example, the micro- and nanoscale biological domains. Light Robotics is beginning to emerge in various applications in biophotonics. Tiny machines allow us to reach places and perform tasks that we can-not do or, otherwise, will be too invasive or lacking sufficient precision. The strength of microrobotics lies in the possibility of controlling multiple machines in parallel or independently [1]. These coordinated movements can allow us to study multiple samples or perform spatially and temporally coordinated stimulation of biological samples.

Inspired by medical robotics, the surgical tools may be scaled down and rein-vented to work at the cellular and even subcellular levels. Understanding cellular dynamics, membrane elasticity, and signaling can help us in diagnosing, designing, and personalizing treatment for debilitating diseases, such as cancer. Realizing these microscopic tools is, thus an interesting and meaningful engineering challenge. In his famous speech, “There’s plenty of room at the bottom,” Richard Feynman, one of the great minds in physics, pondered upon a friend’s suggestion of swallowing a surgeon to perform surgery from within and speculated on how they could be made [2]. Sci-ence fiction films during the 1960s have dramatized various fantastic scenarios along these lines. Now, even the serious optical technology forecasts for the next 100 years are optimistic about realizing such prospects [3]. This brings us back to Feynman’s original question: “How do we make such a tiny mechanism?”

This chapter will cover the challenges and the considerations in making tiny ro-botic machines. There are many ways to actuate tiny machines using chemicals from the surrounding medium or external fields. Light Robotics can use light to both fabri-cate and actuate microrobots but it can also exploit light-based effects and phenom-ena to create functional microrobots. Some of these effects and their respective “light robots” are subsequently discussed. The chapter concludes with an outlook on the various extensions and applicability of the next generation Light Robotics.

2 SCALING OF PHYSICAL EFFECTSHow can we build and actuate tiny robots that are comparable to the size of cells? One major challenge with designing microrobots is that physical effects that are nor-mally ignored at macroscopic scales become significant as we make robots smaller and smaller [4–7]. As the characteristic length L goes down, volume-related proper-ties and effects (∼L3) decay faster compared to surface-related properties and effects (∼L2). Volume related properties and effects include mass, weight, heat capacity, and body forces. Surface related properties and effects include friction, heat transfer, and surface forces. As an example, the weight of an object is negligible at the microscale but adhesion forces become significant. Analysis on the scaling of electrostatic and magnetic forces has been presented by Abbott and coworkers [6] and we show the setup and results in Fig. 9.1. At r < 1 m, the magnetic force dominates gravity while

2672 Scaling of physical effects

this occurs at r < 10−4 m for electrostatic forces. Van der Waals forces also increase at r < 10−7 m. These characteristic lengths are within the typical range when working with biological samples where we would like to apply microrobotics. The significant increase in the magnitude of these forces as the characteristic length of the system decreases tells us that we need to rethink how to implement macroscopic mecha-nisms, such as pumping, rotation of rotors, sliding, and the like.

The scaling of physical laws profoundly impacts the movement of microstruc-tures in a fluid. At microscopic scales, the flow becomes more laminar and viscous forces dominate over inertial effects except at very small timescales. The increase in surface-to-volume ratio also means that small objects have to deal increasingly with thermal fluctuations or Brownian motions [8]. A deeper mathematical treatment on the scaling of physical laws and their effect on micromachines is presented in our cited literature [4,9].

FIGURE 9.1 Scaling of Attractive Forces

(A) Setup to measure electrostatic force where we assume an infinite area for a flat surface. (B) Setup to measure magnetic force where we use a cylindrical magnet. The sphere is at a distance of αr and the force of interaction is measured against gravity. For the simulation, the values used are ρ = 6.72 × 103 kg/m3, U = 100 V, M = 1.1 × 106 A/m.

Image is taken from Abbott J, Nagy Z, Beyeler F, Nelson BJ. Robotics in the small, part I: microbotics. IEEE

Robot Autom Mag 2007;14:92–103 [6] and reproduced with permission from IEEE.

268 CHAPTER 9 Next generation light robotics

Besides the scaling of physical effects, another challenge is how to power and actuate microrobots and how to make them self-contained. One approach is to create miniaturized versions of macroscopic robots. An autonomous microrobot requires a built-in power source to drive its internal mechanisms. Chemical energy stored in a microbattery would supply electricity to an onboard processor and generate the me-chanical, thermal, optical, or other energy to make the robot work. Even if we could engineer miniaturized complex control microcircuits and microbatteries storing suf-ficient energy, we would still have to ask: is all this complexity really necessary? Is there a way to simplify the processes? This requires taking a different approach and to be completely rethinking the microrobotic design. Instead of miniaturizing batter-ies, control circuits, and the micromachinery itself, we can design new microrobotics that can be directly powered by external fields. Instead of embedding or wirelessly sending commands to a self-powered, complex microrobot having its own embedded microprocessor, we can offload the commands and processing to regular computers that are well-suited for these tasks. The same computers can control the required light beams or other external fields to directly power and drive a now much simpler microrobotic embodiment to execute specific tasks at these tiny scales.

3 LIGHT-DRIVEN MICROROBOTS3.1 HARNESSING IDEAS FROM OPTICAL TRAPPING AND MICROMANIPULATIONCompared to other actuation mechanisms like external fields and chemical gradi-ents, optical forces are quite weak ranging only from a few femtonewtons to tens of piconewtons. Nonetheless, optical manipulation offer advantages that makes it at-tractive for actuation of microrobots. The most important is the capability to perform independent and parallel control of a multitude of independent microrobots.

Fig. 9.2 shows the three-dimensional and orchestrated control of multiple mi-crobeads using holographic optical tweezers and counter-propagating GPC-based beams. This functionality can be used to control extended structures, such as micro-robots, through designated optical handles. The previous chapters discuss different optical trapping configurations and how to interface with light. We encourage the reader to go through them and the references therein.

Optical forces can hold microscopic objects having a sufficient refractive index contrast to the trapping medium. This can be inherently problematic for biological samples having low index contrast where increasing the force using more intense trapping beams can have deleterious effects. In such cases, an “optical handle” made of a higher index dielectric material can be employed. These are typically simple glass or polymer microbeads that may be coated to adhere to biological samples. Successful applications of this approach include the calibrated perturbation and characterization of the mechanical properties of living cells and subcellular com-ponents including microtubules, DNA, RNA, and cell membranes [12–14]. These measurements can potentially reveal key cellular properties that can pave the way for

2693 Light-driven microrobots

understanding diseases. For example, it has been found that cellular deformability can be an indicator for malignant transformation and metastatic competence [15]. Another elegant application is the grab-and-drop protocol by Schrems and coworkers [16], where a membrane-associated protein is “grabbed” from the surface of a single cell using a trapped bead and then “dropped” onto a solid-supported lipid bilayer. Moreover, experiments by Kress and coworkers [17] have shown that fabricated mi-crobeads that encapsulate chemoattractants, when dynamically controlled by optical traps, can perform cell stimulation through the chemicals diffusing from the micro-sources. Details on the fabrication of these microsources and their recent biological

FIGURE 9.2

Optical manipulation of a plurality of microbeads using (A–C) holographic optical tweezers where different configurations can be made dynamically using holography. (D) Side and (E) top view of microbeads being held by counter-propagating beams in crystal-like geometries. The orchestrated movement of many particles in 3D space is very suitable for actuating light-driven micro-robots.

Part A–C: Image is from Curtis JE, Koss BA, Grier DG. Dynamic holographic optical tweezers. Opt Commun

2002;207:169–175 [10] and reproduced with permission from Elsevier; Part D and E: Image is from

Glückstad J, Perch-Nielsen I, Dam J, Palima D. Bio-photonics workstation. Proc SPIE 2008;6905:69050A

[11] and reproduced with permission from SPIE.

270 CHAPTER 9 Next generation light robotics

experiments including thermal effects are discussed in Chapter 13. Finally, trapped beads can also be used in photonic force microscopes that perform thermal noise imaging to reveal properties of the material where the probe is placed [18].

Although microspheres work in various applications, using more complex struc-turescan tailor the optical momentum transfer, add new degrees of freedom and open opportunities for realizing novel functionalities. Planar microstructures can be con-veniently made using “subtractive manufacturing” lithographic processes [19,20] while more elaborate 3D structures can be made via “additive manufacturing” direct laser writing (e.g., two-photon fabrication processes). With the availability of pulsed laser sources and UV curable polymers, one can build scanning systems that utilize two-photon absorption processes to perform rapid prototyping of microstructures as depicted in Chapter 6, Fig. 6.1. Complete two-photon fabrication systems are now also commercially available [21]. Chapter 2 discusses the fundamental principles of two-photon photopolymerization.

In an early example by Kawata and coworkers [22], a spring-mass microsys-tem was made by a two-photon process. The spring was stretched by trapping the attached bead to measure its elastic properties. Microscopic gears have also been optically fabricated and rotated by light [23,24]. Chapter 4 presents various heuris-tic designs for light-driven rotating micromachines. For instance, anisotropy in the gear structure can exploit the polarization of light for generating torque [25]. These light-driven rotations can be used to develop micromachines in light robotics. For instance, we can incorporate active control into microfluidics using light-driven ro-tors as light-controlled pumps that generate flow and drag particles. An optically driven paddle wheel [26] exerting shear stress on cells below it can be used to study the cellular response or examine the membrane elasticity. Similarly, optically rotated birefringent materials can control fluid flow and induce morphological changes to living samples, such as axons [27–29]. Results from these birefringent materials and shaped microstructures illustrate that we can augment optical trapping by designing the manipulated object to create a new set of functional microtools for light robot-ics. In the following section, we will discuss some designed structures, created via two-photon processes, which are manipulated by light to perform specific functions.

3.2 TURNING MICROSTRUCTURES INTO FUNCTIONAL MICROROBOTSWith proper design, optically trapped microstructures can work as precision-con-trolled, functional microrobots. These microrobots need two basic elements: (1) a functional load optimized for a specific task and (2) a structural platform that is amenable to optical micromanipulation, which carries the functional load. These two parts are exemplified in Fig. 9.3, which shows a microlens as the functional load held by an optically trapped and manipulated structural platform. The functional load and the trapping handles can be collapsed into the same structure as, for example, when a trapped microsphere is directly used as a microlens for subwavelength laser nanopat-terning [30]. However, decoupling them into two distinct elements means that each can be independently optimized for their respective purposes. A structural platform

2713 Light-driven microrobots

that is optimized for trapping allows the use of microlenses that might be difficult to trap directly (e.g., due to their material or size). Having two distinct design elements, we can create various microrobots that carry optimized functional loads, which may be difficult to trap, as long as they are carried by easily trapped and manipulated structural platforms.

Microrobots can carry other types of microoptical elements and some cases would benefit more from the enhanced control afforded by the structural platform. For example, spherical microlenses may not need angular control but can be essen-tial for asymmetric microoptics, as demonstrated by, for example, the light-driven microrobots called Wave-guided Optical Waveguides (WOWs) [32]. Each of these microrobots carries a waveguide and maneuvered by optical traps through multiple spherical handles. They can redirect incident light from a low numerical aperture objective lens and emit a more tightly confined light at the waveguide exit tip. This allows targeted-light delivery using near-field excitation. It also works for challeng-ing geometries where occlusion is present, as demonstrated by selectively exciting fluorescent beads arranged in odd configurations. Combined with a feedback system, machine vision, and holography, coupling through the waveguides can be done in real-time for a truly automated microrobotics setting [33,34].

Another example of a functional load is the needle-like probe on microrobots designed to perform tasks analogous to atomic force microscopy (AFM), such as force sensing and surface morphology imaging. Unlike AFM, this type of microrobot is not limited to operate in planar geometries but can perform surface imaging of

FIGURE 9.3 Basic Illustration of a Functional Load (a Microlens in This Case) Carried by a Structural Platform That can be Conveniently Controlled by Optical Traps

Various microrobots can be created using functional loads and structural platforms that are optimized for specific tasks.

Image is taken from Bañas A, Vizsnyiczai G, Búzás A, Palima D, Kelemen L, Ormos P, et al. Fabrication

and optical trapping of handling structures for re-configurable microsphere magnifiers. In: Glückstad J,

Andrews DL, Galvez EJ, editors. Proceedings of SPIE vol. 8637; 2013. p. 86370Y [31] and reproduced with

permission from SPIE.

272 CHAPTER 9 Next generation light robotics

three-dimensional objects. Early versions of these 3D scanning probes used natural diatoms [35] and optically assembled structures using rods and microspheres [36]. The probe is held by holographic optical tweezers close to the surface of interest while moving, for example, in a raster-scanning pattern. Analyzing the probe mo-tion can measure forces and reveal surface morphology. Successful scans have been demonstrated for smooth and irregular surfaces like beads and algal structures re-spectively. Recent versions use structural platforms whose shapes are optimized for force clamping. Experiments using this type of probe show a lateral imaging resolu-tion down to ∼200 nm, limited by the size of the tip, while the depth resolution is an impressive 10 nm [37,38]. More theoretical and experimental details are presented in Chapter 3. Incidentally, the momentum change encountered when light travels along a bent waveguide can also enable force clamping [39] using only static illumination. Thus it might be worth exploring in future research how waveguide-induced forces can be used to optimize and enhance optical trapping and manipulation in general.

The next sections deal with light-driven microrobots that use light either to ac-tivate the functional load (e.g., light-driven secondary effects, such as heating and thermal convection) or to exploit optical sensing techniques, for example, surface-enhanced Raman and temperature-dependent fluorescence. We also discuss the fab-rication protocols required by these additional features, for example, to incorporate metallic structures into light-driven microrobotics. We complete this chapter by show-ing experimental demonstrations of this next generation of light-driven microtools.

4 MICROROBOTS WITH VARIOUS FUNCTIONAL LOADSDifferent microrobots can be created using different functional loads as illustrated by various examples in the different chapters of this book. We briefly discuss some of them later, for illustration, and present other unique examples. Several of the ex-amples use functional loads containing metallic structures and so we will start with a short survey of the various potential applications of metallic structures.

4.1 METALLIC STRUCTURES IN MICROFLUIDICSThe generation of heat in microfluidics can initiate convection that can be used to mix things and facilitate chemical reactions, or other thermal effects, such as micro-bubble generation. However, it is not straightforward to generate heat since lab-on-chip devices made from PDMS, PMMA, and the like, including the applied liquid medium, normally do not absorb the laser wavelengths used in biophotonics. It is sometimes necessary to use pulsed laser sources to reach the required temperatures. In addition, a precise spatial control of the heat source is also a challenge.

Metallic particles and light-absorbing structures can convert optical energy to heat to lower the laser power needed in heat generation. Heating nanoparticles with a laser can control optofluidic flow by cyclic conversion of liquid to vapor and back to liquid droplets. As the droplets coalesce with the main bulk of liquid, the liquid-air

2734 Microrobots with various functional loads

interface also moves—as do the particles [40]. However, precisely controlling nanoparticle positions is challenging [41]. Alternately, laser-induced heating of thin metallic layers embedded in microfluidic channels can generate bubbles, which can then be used as pumping function [42]. Others have used surface plasmons in fluid mixing [43] and plasmon-based trapping has also been demonstrated [44]. Metallic films are shown to improve Förster resonance energy transfer (FRET) microscopy by increasing its applicability up to 100 nm [45]. Typically, FRET is limited to 10 nm separation between the donor and acceptor molecules.

Metallic nanoparticles arranged in an array can serve as efficient nanoheat sources us-ing localized surface plasmons [46,47]. They can induce fluid flow as shown by numeri-cal simulations [48] and experimentally demonstrated in optofluidic applications [49]. A comprehensive review on the integration of plasmonics with microfluidics including surface-enhanced Raman spectroscopy in lab-on-chip settings is presented in [50].

Integrating metallic structures in microfluidics presents an elegant solution to fluid control and sample analysis. In the next section, we will present light-driven microrobotics that incorporate metal-based functionalities together with enhanced spatial and temporal control from optical micromanipulation. We also discuss a few other exciting examples from the literature.

4.2 HOLLOW MICROROBOTS FOR MATERIAL TRANSPORTControlled transport of cargo has potential applications in drug delivery. For the light-driven microrobots that we will present here, we fabricated a hollow micro-robotic architecture including embedded metallic structures. A spout at the anterior end works for loading and unloading cargo in and out of the hollow microrobot. Four spherical handles with 8 µm diameter are added to the structure for optical ma-nipulation. This type of microrobot has been made using a commercial two-photon fabrication setup (Nanoscribe Photonic Professional, Nanoscribe GmbH, Germany) and their proprietary photoresist (IP-L 780). The fabrication setup uses a 780 nm pulsed laser (100 fs pulses, 80 MHz repetition rate and ∼ 140 mW average power) to print voxels via two-photon polymerization at the focus of a high numerical aperture microscope objective. The photoresist is placed on a glass substrate by drop casting and then placed on a piezo-controlled microscope stage. Cartesian coordinates rep-resenting the desired structure are fed into the software that controls the laser power and stage position during fabrication. One can define coordinates, for example, using CAD software and slicing the resulting volume data for a layer-by-layer fabrication. Here we have chosen the shape of the microrobot based on the surface of revolution of a teardrop curve [51,52] and calculated the coordinates using parametric equations

θ

θ θ φ

θ θ φ

=

=

+

=

+

x A

y B r

z C r

cos

sin sin1

2cos

sin sin1

2sin

m

m

(9.1)

x=A cos θy=B sin θ sinm12θ+rcosφz=C sin θ sinm12θ+rsinφ

274 CHAPTER 9 Next generation light robotics

where we set A = 20 µm, B = 12 µm, and C = 8 µm. The radius of the spout is r = 3 µm and the parameter m = 5 sets the length of the taper from the main hollow body to the spout. The hollow design anticipates an application where the microrobot picks up and carries cargo that it can subsequently inject into living biological samples.

We left an 8 µm diameter hole on top as entrance port for depositing a thin metal layer inside the microrobot by vapor deposition. A mask with a matching hole photo-polymerized over the structure shields the rest of the structure during the deposition. After two-photon exposure, the microrobots are submerged into an isopropyl alco-hol bath to remove unpolymerized resists (Fig. 9.4A). This is followed by electron physical vapor deposition of the metal layer. The deposition process starts with a

FIGURE 9.4 Fabrication Protocol for Metal-Embedded Microrobot for Material Transport

(A) The basic structure is fabricated using two-photon polymerization. A mask is fabricated on top of the microrobot to expose only the region where the metal layer is to be deposited. (B) The metal layer is deposited via electron beam vapor deposition starting with a 1 nm adhesion layer made of titanium and followed by 5 nm of gold layer. (C) Scanning electron micrograph of the fabricated structure. (D) The microrobots are collected interactively using a fine glass capillary tube and later transferred to a cuvette.

Image is from Villangca MJ, Palima D, Bañas AR, Glückstad J. Light-driven micro-tool equipped with a

syringe function. Light Sci Appl 2016;5:e16148 [53] and reproduced with permission from NPG.

2754 Microrobots with various functional loads

1 nm layer of titanium as adhesion layer and then followed by 5 nm layer of gold (Fig. 9.4B). The developed and metal-coated structure is shown in Fig. 9.4C.

The finished microrobots are removed from the substrate using a fine glass capil-lary tube attached to a microliter syringe. Motorized actuators control the syringe (Figure 9.4D) to collect selected microrobots and to transfer them into a cytometry cuvette where optical manipulation experiments can be performed. The trapping me-dium consists of 0.5% Tween 80 and 10% ethanol in deionized water.

After collection, optical manipulation is performed on our Biophotonics Work-Station [11,54]. Counter-propagating beams trap and hold the spherical handles to move each microrobot with six-degrees of freedom. Another trapping beam is used to heat the metal layer inside. Previous experiments on nanodot arrays have revealed that bubble formation occurs at temperatures between 220 and 240°C [55]. Thus, when the metal layer is sufficiently heated, a bubble and a strong convection current are formed. To demonstrate loading and spatial control, a microrobot is optically ma-nipulated and brought close to its cargo (e.g., 2 µm silica beads) as shown in Fig. 9.5. The generated convection current is strong enough to pull in silica beads inside the hollow body of the microrobot. The spatial control from the optical traps allows us to pick up the cargo scattered inside the cuvette.

The flow speed of the particle is measured using a feature-tracking algorithm at the minimum laser power to initiate convection current (around 17 mW) as shown in Fig. 9.6. The tracer particle moves slowly outside the microrobot but reaches around 10 µm/s once inside the spout. The particle slows down as the channel widens but

FIGURE 9.5 Scattered Silica Beads (φ = 2 µm) are Being Collected Using the Thermal Convection Current Generated by Heating the Metal Layer Inside the Microrobot

The position of each microrobot is controlled by four counter-propagating beams each targeting a spherical trapping handle and a fifth beam is designated to act as heating beam.

276 CHAPTER 9 Next generation light robotics

suddenly gains speed near the metallic structure. Two convection currents are at work here—the natural convection due to the temperature gradient and so-called Maran-goni convection. The temperature gradient from the bottom to the top surface of the bubble causes a surface tension gradient and Marangoni convection occurs. Maran-goni convection has been used in pattern formation by utilizing the current flow to collect particles and depositing them on a substrate [56,57] and has also been used in mass transfer of dissolved molecules [58]. Marangoni convection can be very strong reaching as high as 0.3 m/s at a liquid–gas interface [57]. This is significantly stron-ger than when using optical forces to drag particles. Moreover, unlike conventional optical trapping that requires index contrast between the medium and the object, this method is not dependent on the optical properties of the object. Hence, the experi-mental result represents an alternative way to interact in a microscopic environment that overcomes fundamental limitations of optical trapping.

Marangoni convection has been used to control fluid flow in microfluidics by using carefully placed heating elements [59]. The use of a microrobot as vehicle for the heating element bestows spatial control on the location of the heat source and the microrobot can be used to transport cargo. Using the generated microbubble as pump for the microro-bot’s functional load—essentially a syringe—the loaded cargo can be ejected by moving the heating beam across the body of the microrobot as shown in Fig. 9.7. This structure-mediated approach also prevents unwanted illumination of light-sensitive samples.

FIGURE 9.6 The Flow Speed of the Thermal Current is Measured Using a Tracer Particle

A feature tracking algorithm is used to track the particle. The two plots represent the tracer’s speed outside and inside the microrobot. The gap is due to the inability of the algorithm to identify the tracer at the dark outline of the microrobot.

Reproduced with permission from NPG.

2774 Microrobots with various functional loads

Optimizing microrobots for their loading and unloading capabilities can have potential uses in the area of drug delivery. The ejection of cargo can be optimized by investigating the underlying mechanisms further. The following mechanisms may be involved: (1) thermocapillary bubble migration [60], (2) change in flow patterns due to change in direction of thermal gradient [61], and (3) reversal of Marangoni con-vection due to presence of many particles [57]. First, in thermocapillary bubble mi-gration, the bubble is strongly attracted to the heat source, which is the laser beam in this case. This attraction can be two orders of magnitude larger compared to optical trapping forces. The images show that moving the heating beam causes the bubble to shift, which can effectively work as a piston pump. Second, moving the heating beam alters the thermal gradient, which perturbs the flow around the microbubble. Third, the reversal of the Marangoni convection has been observed in numerical simulations when there are many particles.

4.3 MICROROBOTS FOR CHEMICAL SENSING: PROBE FOR SURFACE-ENHANCED RAMAN SPECTROSCOPYRaman spectroscopy reveals the unique molecular fingerprint of different samples and, thus can be used to identify samples. A small amount of sample is needed and the measurement and analysis can be integrated into a microfluidic setup. Interest-ingly, trapping beams can also be used as the excitation beams for Raman spectros-copy in a configuration as so-called “Raman tweezers.” This configuration again uses the functional load as the trapping handles as the sample is simultaneously trapped and analyzed. This has been used to characterize trapped cells and particles [62–64]. One practical limitation of Raman spectroscopy is that the Raman signal is very

FIGURE 9.7 By Spatially Varying the Position of the Heating Beam, the Microrobot can also Eject Particles

(A,B) This functionality mimics the action of a syringe and can have applications in controlled drug delivery.

Reproduced with permission from NPG.

278 CHAPTER 9 Next generation light robotics

weak. One approach is to use plasmonics for field enhancements, such as in surface-enhanced [65] and tip-enhanced [66,67] Raman spectroscopy (respectively SERS and TERS). Metal surfaces can be added to microfluidic channels in order to perform on-chip SERS while TERS can be done on an atomic force microscope (AFM) tip. The field enhancement from the above techniques increases the signal of the Raman spectra although they lack spatial control due to their geometry.

Optically manipulated microrobots offer enhanced spatial control and the previ-ous section shows that such devices can be fabricated to contain metallic nanofilms. Other methodologies can be used to deposit the metallic structure into the micro-robots. Vizsnyiczai and coworkers [68] made SU-8 microrobots using two-photon fabrication and subsequently used the photoreduction of a silver nitrate solution to deposit a rough metal surface onto the fabricated structures. A 532 nm laser beam aimed at the sensing tip of the microrobot was used to perform photoreduction where solid silver was deposited at the illuminated area. A fabricated shield minimized deposits on the rest of the structure. The metallic surface produced on the tip of the microrobot was grainy and, hence, suitable for localized surface-enhanced Raman spectroscopy. The roughness affects the transmission of the 532 nm laser and could be selected by monitoring the transmitted beam (Fig. 9.8). This microrobot sepa-rates its functional load from the structural platform having spherical trapping han-dles, thus allowing the use of different trapping and Raman excitation beams, as shown in Fig. 9.8E. This microrobot can therefore function as a three-dimensional SERS probe. The experimental results show that the SERS probe can achieve a sig-nificant improvement in the measured Raman spectra of emodin, an anticancer drug, compared to bulk measurements and results when using the microrobots without metal coatings. More details are available in Chapter 6.

FIGURE 9.8 Surface-Enhanced Raman Probes

The sensing tip of the microrobot is coated with silver using photoreduction. The distribution of the grains of silver affects the transmission of the illuminating beam; different distributions shown are for (A) 20%, (B) 30%, (C) 50%, (D) 80% transmissions. (E) The microrobot is held by two trapping beams and a separate beam is used to excite the molecule of interest (e.g., emodin) for SERS measurements.

Image is taken from Vizsnyiczai G, Lestyán T, Joniova J, Aekbote B, Strejcková A, Ormos P, et al. Optically

trapped surface-enhanced raman probes prepared by silver photo-reduction to 3D microstructures. Langmuir

2015;31:10087–10093 [68] and reproduced with permission from ACS.

2794 Microrobots with various functional loads

4.4 MICROROBOTS FOR TEMPERATURE SENSINGThe application of different fabrication methodologies can tailor the functional load to create microrobots having different kinds of functionalities. A metallic nanolayer inside a microrobot cavity was generating convection for the syringe-like functional load discussed earlier. Another thermal application of microrobots, or nanorobots as the authors call them, uses dye-doped functional loads to measure temperature [69]. The fabrication process, shown in Fig. 9.9, uses two-photon polymerization of a mixture of SU-8 and Rhodamine B to polymerize doped structures. Rhodamine B is typically used in biological staining and as dye laser gain medium. The fluores-cence from Rhodamine B is temperature-dependent and can be used, for example, to measure the temperature distribution in solution (see e.g., calibration data presented in Fig. 13.2, Chapter 13). Doping microstructures with Rhodamine B thus creates a controllable, temperature-sensing microrobot.

To demonstrate distributed temperature sensing, nanowires can be embedded into nanorobots by fabricating them around silicon nanowires (150 nm thickness, ther-mal conductivity 148 W/m·K) by dispersing them in the photoresist mixture. The trapping chamber is directly built around the fabricated nanorobots using PDMS (Fig. 9.9A). During experiments, the nanorobots are dislodged from the surface by ablating the supporting structure with a pulsed laser.

Mechanical manipulation is done using holographic optical tweezers and the re-sults shown in the experiments of Fukuda and coworkers [69] indicate that it can

FIGURE 9.9

(A) Fabrication of a temperature sensor. A nanorobot is made from a mixture of SU-8 and Rhodamine B where the silicon nanowires are also dispersed after an initial spin-coating. Two-photon fabrication happens at the vicinity of a nanowire which is used as a thermal probe. The trapping setup is built around the nanorobots where the samples are later introduced. The nanorobots are released from the substrate by ablating their supporting micropillars. (B) A schematic of a nanorobot with a silicon nanowire as thermal probe.

Image is taken from Fukada S, Onda K, Maruyama H, Masuda T, Arai F. 3D fabrication and manipulation

of hybrid nanorobots by laser. 2013 IEEE Int Conf Robot Autom, IEEE; 2013, p. 2594–2599 [69] and

reproduced with permission from IEEE.

280 CHAPTER 9 Next generation light robotics

indeed be used for temperature measurements. Fig. 9.10B–D shows fluorescence images when varying the homogeneous sample temperature (B–D). Fig. 9.10E shows inhomogeneous temperature measurements along the doped polymer when the nanowire tip is heated by laser illumination.

The fabrication process described here can be adapted to create new microrobots based on different functional loads. For example, in Chapter 7, Arai et al. are using a similar fabrication process but use carbon nanotubes to exploit their large photother-mal effect to puncture cell membranes. One can also consider other functional loads, for example, the LbNO3 nanowires in Chapter 8 can potentially be optimized for sec-ond harmonic generation. Finally, the body of literature on nanowire photonics [70] and dye-doped polymers [71] is a rich resource waiting to be tapped for realizing new functional loads to advance the emerging field of light robotics.

5 CONCLUSION AND OUTLOOKLight robotics represents an extension of optical manipulation by merging with other disciplines to create light-driven microrobots that carry functional loads on struc-tures optimized for optical trapping to enable new functionalities. Microfabrication plays an important role both in optimizing the support structures and the functional loads. We have presented several polymer microrobots that incorporate other materi-als for added functionalities. The photothermal effect in metals can create hydrody-namic effects that can be potentially much stronger than optical forces and are not

FIGURE 9.10

(A) Temperature sensing is demonstrated by heating the silicon nanowire with a focused laser beam. Temperature reading is made at three locations along the nanowire. The fluorescence of Rhodamine B is temperature-dependent. The intensity decays as temperature increase. Measurement of the fluorescence intensity for (B) 308 K, (C) 312 K, and (D) 314 K are used for calibration. (E) The experimental results from three measurements along the nanowire.

Image taken from Fukada S, Onda K, Maruyama H, Masuda T, Arai F. 3D fabrication and manipulation

of hybrid nanorobots by laser. 2013 IEEE Int Conf Robot Autom, IEEE; 2013, p. 2594–2599 [69] and

reproduced with permission from IEEE.

281References

limited by the refractive index contrast. They can also be used to enhance signals in Raman spectroscopy and fluorescence [72]. Microrobots carrying these metal-lic structures provide high spatial and temporal control. Tailoring the design of the embedded metallic structures can take advantage of plasmonics to enhance fields, improve light-matter interaction and provide wavelength selectivity. For example, a plasmonic nanomotor (radius: 100 nm) has been shown to drive a silica microdisk (2.2 × 2.2 µm2) [73].

Functional loads can exploit various optical effects in metals and other materials. Nanowires can have strong photothermal effects and other interesting nonlinear opti-cal response functions. Chapter 8 discusses the optimization of harmonic generation in lithium niobate LiNbO3 nanowires. Frequency generation has been demonstrated in optically trapped potassium niobate nanowires with basic mechanical control [74]. Incorporating nanowires as functional loads in light-driven microrobotics improves maneuverability and the independent optimization of trapping and functional load can prove useful in novel microrobotic probes and sensors.

Understanding the inner workings of biological processes is essential for devel-oping new and improved tools to diagnose and treat various diseases. Optical forces have been already used to show that cellular deformability can be used as cancer marker [15]. The independent yet parallel control of multifunctional microrobots can be an attractive feature for exploring biology with light robotics. Various microrobots can be designed to exert coordinated stimuli and sense the response of subjects. They can also provide an alternative approach to single cell transfection [75–77] where, for example, optoporation and loading mechanisms of plasmid-coated microparticles can be built-in. Needle-like structures can be integrated for drug delivery [78]. The flexibility of the fabrication methods allows rapid prototyping of microrobots for exploring various functions.

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CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00010-0Copyright © 2017 Elsevier Ltd. All rights reserved.

Duncan Casey*, Douglas Wylie**, Mark Neil***Centre for Functional Nanomaterials, University of Bristol, United Kingdom;

**Imperial College London, London, United Kingdom

CHAPTER OUTLINE

1 Introduction and Overview ................................................................................. 2872 Direct Optically Triggered Membrane Effects ...................................................... 289

2.1 Theory and Background ..................................................................2902.2 Choosing Your Methodology .............................................................292

3 Secondary Target Techniques ............................................................................ 2983.1 Gold .............................................................................................2993.2 Thin Film Deposition ......................................................................302

4 Conclusions ...................................................................................................... 307References .............................................................................................................. 307

1 INTRODUCTION AND OVERVIEWThe developing field of light robotics offers a new suite of technologies with which to investigate the processes of life at its most fundamental level. Optics offers a wide variety of techniques to deliver control at the micron or submicron level with minimal invasion and potentially at high throughput, particularly compared to bulk mechani-cal or chemical methods. Furthermore, recent advances in microscopy, lasers, and control optics have made such techniques convenient and accessible to life-scientists in the laboratory. Cellular-scale, mechanically- and chemically-configurable probes can be controlled using contact-free optical techniques, meaning that for the first time the experiment can travel to the biology rather than vice versa. This provides a unique opportunity to investigate the functions and behaviors of individual cells in their na-tive culture, minimizing or eliminating altogether the confounding effects observed through techniques such as trypsinization [1]. Furthermore, it offers the resolution to perform these studies at the single- or even subcellular level, permitting genu-inely quantitative biology experiments able to tease apart the differences between subpopulations within a bulk cell culture or biopsy sample [2].

Optical techniques and microtools for subcellular delivery and sampling

10

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This chapter will focus upon the production and application of light robotics tools designed to interact directly with the cell membrane, disrupting it to allow access to the complex material within (illustrated schematically in Fig. 10.1). The cell membrane has evolved over billions of years to isolate and contain the sensitive, electron-rich macromolecules that underpin life’s processes, protecting them from environmental damage and regulating the flux of nutrients and waste through the sys-tem. In doing so, however, it poses a major challenge to the experimental researcher as it effectively restricts the flow of material both into and out of the cell. While many techniques have been developed to rupture it, the majority are somewhat crude and typically lead to uncontrolled lysis of hundreds or thousands of cells within an affected area. This imposes severe limits on the experiments possible, swamping information about subpopulations within the mass of data, but also means that such

FIGURE 10.1 A Schematic Representation of the Most Common Optical Techniques for the Disruption of Cell Membranes Using Light Robotic Tools

(A) Direct pulsed laser-induced breakdown (LIB) of water, generating a substantial plasma cavitation bubble and subsequent shockwave that damages all cells in the vicinity. (B) Pulsed or short-wavelength laser focused on the membrane itself, generating a low-density plasma which can maintain a micron-sized pore across the structure. (C) Shows thermal fluctuations generated in the lipid barrier by direct irradiation with a continuous-wave beam, while a closely-related effect can be generated by (D) focusing a beam that is not absorbed by biological tissue onto a target surface or microstructure, thus greatly reducing the cell’s effective exposure . The energies absorbed by the cell can similarly be produced by (E–F) targeting micro- or nanoparticles (shown as black circles) which may be independently steered using orthogonal techniques, such as magnetism. These may be used for (E) direct membrane interactions or for (F) relatively low-energy laser-induced breakdown in water.

2892 Direct optically triggered membrane effects

experiments can only ever be snapshots of a given cell population’s state. Each mea-surement must be made using a different group of cells, and even those derived from a clonal suspension have been observed to display significant differences in behav-ior: for example, Schubert et al. have recently demonstrated prostate-specific antigen levels displaying 100-fold changes in intracellular concentration among neighboring LNCaP cells [3].

The ideal experimental tool, then, allows selective and nondestructive poration (or triggered endo-/exocytosis) of an individual cell within its native, three-dimensional environment. In this way, individual cells can be repeatedly probed and analyzed, providing real-time feedback about cellular response(s) to stimuli. Light robotics tools, coupling precise three-dimensional control with tunable surface properties, provide the ideal combination of properties to deliver stimuli, abstract material, or read out optical information generated from cells of interest.

While this “complete,” all-in-one biochemical workbench remains a prospect for the future, significant strides have been made toward it. In this chapter, the evolution of light robotics tools for single cell biochemistry and biophysics will be discussed, focusing upon the development of techniques for controllably disrupting the cell membrane’s structure and function in as gentle and precise manner as possible. The chapter will be split into two major sections: one describing the direct interaction of laser light with cell membranes, and the second highlighting recent research into the interplay between laser light and membranes coupled to secondary targets. These secondary targets are of particular interest, offering novel opportunities around tuned surface coatings and microstructures while greatly minimizing the required incident laser flux.

2 DIRECT OPTICALLY TRIGGERED MEMBRANE EFFECTSThe interactions between coherent light and biological materials were explored from a relatively early stage in the development of laser techniques. Tsukakoshi et al. demonstrated the reversible poration of individual cells for DNA transfection in 1984, utilizing a 355 nm 5 ns laser pulse to induce a rapidly healing pore wide enough to admit DNA plasmids [4]. These early attempts were fairly crude: success rates were low, largely due to the damage done to target cells during the poration process. Importantly, however, they demonstrated that biological membranes could be manipulated in a contact-free process that was as versatile and adaptable as the optical bench used to mediate the effects.

While many techniques were and are still available to disrupt membranes, osmotic, thermal, electrical or chemical techniques such as detergent solubilization each present a wide area of effect. Although perfectly suitable for many applications, this is further complicated by their often uneven distribution: diffusion, thermal transfer, and edge-effects means that cells typically receive a range of differing exposures, over-stressing some cells while appearing ineffective to others. For the first time, laser techniques offered the potential to lyse and selectively porate the membranes of small numbers

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of target cells without their physical isolation. Even at its crudest, using a single laser beam steered by manually-controlled mirrors, individual cells may be effectively tar-geted in suspension or culture. When coupled to advanced beam-shaping techniques such as holographic spatial light modulators, multiple beams can be generated and moved in parallel, providing a true light robotic platform for membrane disruption (Chapter 2 discusses generating multiple beams holographically for trapping).

2.1 THEORY AND BACKGROUND2.1.1 Mechanisms of actionThroughout the development of light-mediated membrane disruption, a range of la-ser wavelengths, powers and beam conditions have been utilized depending on the outcome required and the state of the art instrumentation of the time. As a result, a number of distinct poration modalities have been observed: the interactions between a membrane and a short-wavelength pulsed laser with a very high power density are very different to those with infrared, continuous wave sources, as shown in Fig. 10.1. While the desired result is often very similar, this flexibility allows the fine-tuning of a protocol to reflect the individual requirements of the application.

The two main processes by which a laser disrupts a membrane can be split be-tween low-density plasma formation and thermal fluctuation. Every approach in-volves a degree of thermal contribution, but there is a minimum energy density re-quirement for plasma-based processes whose effects overwhelm and dominate the thermal fluctuations they also trigger. However, plasma formation can again be sub-divided between processes causing laser-induced breakdown of water or other mate-rials in the surrounding medium to create a cavitation shockwave (where an area of effect is required to disrupt a number of cells), and generation of a single pore in one target cell via direct targeting.

Plasmas are created by stripping the electrons from atomic nuclei to create high energy, low density bubbles capable of disrupting almost any material they encoun-ter. However, these bubbles are extremely short-lived, decaying almost immediately upon cessation of illumination, while their energy requirements keep them confined within the focal volume of the laser beam. These same energy requirements limit the effects to either short-wavelength light sources in the blue-ultraviolet range, or to extremely high flux, pulsed sources at longer wavelengths which rely upon multiphoton effects to achieve the same goal (discussed further in Section 2.2.2.1).

By contrast, longer wavelength and most continuous wave sources are unable to reach the flux levels required to maintain plasma, or lack photons of sufficient energy to trigger electron release. These rely upon direct heating effects to destabilize their target membranes, using localized heat gradients to trigger thermal fluctuation within the lipid bilayer. Cell membranes are self-assembled detergent structures, held to-gether by the high energetic cost of exposing the lipids’ hydrophobic chain regions to the surrounding water (Fig. 10.2). Proteins embedded in this matrix use the same effect to maintain their structure and depth within the bilayer: most transmembrane proteins feature an amphiphilic region comprising a hydrophobic barrel topped and

2912 Direct optically triggered membrane effects

tailed with hydrophilic regions, while many membrane-associated proteins simply couple themselves to specific lipid moieties as anchors [5]. However, the lack of co-valent interactions between the constituents mean that components can flow in what is essentially a two-dimensional fluid. Furthermore, it is a fluid capable of adopting complex geometries: while the flat, lamellar structure is the most familiar, lipids are capable of reversibly switching between a range of highly curved structures featur-ing stalks, pores, and channels of their own [6]. These structures are most obvious in organelles like the Golgi apparatus and the endoplasmic reticulum, where curvature and the high surface area that it brings are intrinsic to the structures’ biological roles (Fig. 11.1, Chapter 11) [7]. However, the cell also actively maintains its membranes in metastable states that require only a few kBT of energy to transform, in order to

FIGURE 10.2 Lipid Membrane Structure at the Nanoscale, Schematically Illustrating the Formation of the Range of Lipid Membrane Bodies Seen In Vivo

Individual lipid molecules, exemplified by 1,2-dilauroyl-sn-glycero-3-phosphorylcholine (A), typically consist of a hydrophilic head-group coupled to one or more hydrophobic alkyl tails. In water, these molecules cluster together to shield the hydrophobic regions, forming bilayer structures that act as a two-dimensional film of oil with a depth of approximately 5 nm (B). Electrostatic interactions between the bilayer head-groups (and other forces mediated by proteins etc.) arrange these films into stacks separated by water channels of sub-nm dimensions, which may come into contact with one another during thermal fluctuations or other perturbations (C). Under the correct circumstances, these contact points can become stalks (D) [9], which can lead to a range of more exotic structures. Microtubules, such as those shown in (E) form the basis of structures, such as fusion pores and membrane filaments, while small micelles (F) are extensively used in exocytosis and neurotransmitter signaling. More highly curved structures, such as the cubic phases (G) [6] arise in complex, membrane-based organelles, such as the Endoplasmic Reticulum and Golgi Apparatus (Fig. 10.1, Chapter 11 for details).

Panel G is adapted from Shearman et al. Inverse lyotropic phases of lipids and

membrane curvature. J Phys Condens Matter 2006;18: S1105–S1124 IOP Publishing.

Reproduced with permission. All rights reserved.

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facilitate processes such as endocytosis which require highly curved membrane in-termediates [8]. This energy is quite straightforwardly accessible with a laser source of only modest power: targeting leads to thermal defects forming within the struc-ture, forming channels through which material can flow both into and out of the cell.

A number of optoporation approaches and methodologies are described later, along with relevant examples of their practical implementation. However, a compre-hensive exploration of the mechanistic underpinnings and history of each single cell-poration and transfection technique cannot be delivered in depth in a chapter as broad in scope as this: the interested reader is directed to a number of excellent reviews on the topic available elsewhere [10,11].

2.2 CHOOSING YOUR METHODOLOGYEach approach offers its own mixture of advantages and disadvantages, meaning that the mechanism of optoporation or optical lysis should be considered carefully before the start of an investigation. Short wavelength sources suitable for plasma bubble generation are very strongly absorbed by biological material, meaning that simple, low power systems can trigger poration in target cells. However, the lipids of the membrane are transparent to all but the hardest, shortest-wavelength sources, and the vast majority of the absorption is thus by the proteins and nucleic acids within the cell. These are normally the analytes of interest, but their absorption of high energy photons can induce a range of photochemical effects that may impair their subsequent function or generate artefacts in the data so generated.

By contrast, thermal effects are typically gentler, but far more widespread in their influence. The dynamic, fluid nature of the lipid membrane means that energized par-ticles rapidly diffuse away from the center of heating, causing temperature hotspots to rapidly equilibrate around the cell and then away into the surrounding medium. As a result, it is challenging to selectively porate individual cells using direct thermal techniques, and care must be taken to avoid denaturing biomolecules in the region of interest. However, this same effect means that the energies experienced by off-target proteins etc. throughout the cell are commensurately lower.

2.2.1 Plasma shockwave generationIf lysis or disruption across an area of effect of hundreds of microns is the desired out-come, water cavitation may be an appropriate approach. In this technique, a short-lived plasma bubble is generated in the cells’ buffer or growth medium using a pulsed laser [12]. This bubble expands and collapses rapidly, creating a swiftly-transduced pressure wave which decays as it spreads. By definition, this means that cells closest to the focal region of the laser experience the greatest shearing forces and are typically damaged ir-reparably, while those further from the epicenter experience much less disruption. The shockwave decays sharply, proportionally to the cube of the distance from the focal point, which means that effects are limited to a few hundreds of micrometers. While this volume may contain dozens of cells, it is a relatively simple technique to employ and can be adapted for a range of lysis and poration experiments.

2932 Direct optically triggered membrane effects

Through careful positioning of the pulsed beam, a range of effects may be caused. Cells near the epicenter will be lysed to their constituent molecules, gen-erating a blizzard of proteins and related cell detritus [13]. Those furthest from the cavitation bubble will reversibly and transiently porate as the cell absorbs the incident shear forces [14]. One recently reported technique goes a step further, and uses edge-effects within a culture environment to induce asymmetric bubble collapse leading to microjetting [15], similar to that observed in ultrasonic experi-ments (Fig. 10.3).

However, potentially the most useful application of the technique lies in the ef-fects on those cells in the middle ground: the technique has proved to be a convenient route to the isolation of intact cellular organelles [16]. In these cases, the outer cell membrane is obliterated to release the cell’s cytosolic content, but the forces are insufficient to substantially damage the interior structures.

While relatively straightforward to set up and implement, these techniques have major limitations. The magnitude of the effect is exquisitely sensitive to the target structure’s proximity to the laser’s focal point, making reproducibility between labo-ratories a serious challenge. Cells from biopsy or culture rarely grow in neat con-centric rings, meaning that in most instances there will be a spectrum of responses that are likely to introduce confounding variables. While it can be used to produce time-lapse data from one culture environment, moving the laser target in between analyses to a fresh region of cells, it is difficult to eliminate the effects of a cell’s

FIGURE 10.3

Experimental (A) and simulated (B) responses of a cell to an optically generated cavitation bubble, demonstrating selective membrane pore formation. While this technique irreparably damaged the cells in question, it demonstrates how elegant combinations of cell chamber microstructuring and optical poration techniques can deliver single or even subcellular specificity.Reproduced from Li et al. Single cell membrane poration by bubble-induced microjets in a microfluidic chip.

Lab Chip 2013;13:1144 with permission from The Royal Society of Chemistry [15].

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lysis products on those around them. As such, the approach is often best utilized as a total lysis technique, to violently disrupt the structures of cells and organelles within a defined and restricted target volume.

2.2.1.1 Microfluidic isolation, capture, and downstream analysisEach cavitation-bubble approach causes disruption to an area around the laser focal spot, inducing an inevitable range of responses across any given volume. As a result, a number of microfluidic techniques have been developed to lyse or isolate cells of interest in as gentle a manner as possible, to minimize any possible influence from the cell’s detachment on either the target or those cells around them. A full review of microfluidic techniques for single cell analysis is beyond the scope of this chapter and worthy of a textbook in its own right: the interested reader is directed to a recent, con-cise introduction to the topic presented by Lo and Yao [17]. However, the vast major-ity require that the cells are detached from culture before any manipulation or analysis can begin: two techniques which stand out from this field are highlighted below.

Sarkar et al. have developed a microfluidic probe system capable of hydrody-namically confining a detergent suspension to sufficiently narrow a region that an individual cell may be lysed and analyzed without damaging those around it, even in a confluent monolayer [18]. The confinement is sufficient that both the detergent mixture and the lysate it contains after exposure may be recaptured and analyzed. While intrinsically a low throughput technique, the picoliter volumes involved yield relatively high analyte concentrations, making this an impressive display of micro-fluidic control.

Salazar et al. have gone a conceptual step further, demonstrating that cells grown on a microstructured substrate can be isolated while retaining their extracellular anchors, by developing a photolabile system of islands or “micropallets” produced from biocompatible SU-8 [19]. These micropallets, separated from each other by barriers of trapped air, kept cells alive in culture but were ejected from their host array by means of a laser pulse. However, the pallets were sufficient to protect the cells from laser damage and once captured, could be moved to a separate chamber for analysis or used as the basis for a seed colony.

2.2.2 Direct cell targetingThese microfluidic approaches offer the opportunity to analyze cells of interest in their native culture and eliminate (or at least minimize) artefacts due to trypsinization or related mechanical stresses [1]. However, each technique is intrinsically limited to a relatively low throughput, and the cell of interest is removed from its environ-ment (and potentially destroyed) in the process of sampling. This means that repeat experiments or longer-term observation of cells of interest are essentially impossible. A more delicate application of light robotic principles than has been outlined so far can deliver direct interactions with regions of specific cell membrane, even among a confluent monolayer of neighbors. This process can be tuned to develop membrane pores and channels with lifetimes from seconds to minutes, although cell survival is inversely proportional to exposure time.

2952 Direct optically triggered membrane effects

The approach was pioneered by Tsukakoshi et al. in the mid-1980s [4]: their experiments using a pulsed 355 nm laser source demonstrated the formation of mem-brane pores of single-micron dimensions within the laser focal cross-section. These pores could be generated from a single pulse of the laser, providing energy of around 1 mJ, and healed over the course of half a second after irradiation. While a powerful demonstration of the new technique, the pores formed were extremely disrupting to the target cells: pores were of 2–3 µm diameter in cells of 10–12 µm width, which means that a substantial fraction of the cell was affected by the process. As a re-sult, success rates were low: even the most optimized conditions yielded only ≈10% effective transfection and survival.

What the technique did offer, however, was the potential of genuinely high-throughput transfection with single-cell precision. In these experiments beam steer-ing was performed using manually-controlled mirrors, but the combination of more advanced optics coupled with the high speed of the poration itself means that the system could in theory process many thousands of cells in a matter of minutes. Fast-switching spatial light modulators and related technologies [20,21] can holographi-cally generate multiple, individually-addressable diffraction spots from a single input source, massively parallelizing the process.

As alluded to in Section 2.2, the extremely high extinction coefficients of short wavelengths in biological tissue means that very low beam intensities can be used to effectively porate membranes: almost every incident photon will be captured by the cell and its energy utilized. The experiments of Tsukakoshi [4] used a third-harmonic beam generated from an Nd:YAG laser source [22], but Paterson et al. in the labora-tories of Kishan Dholakia at the University of St Andrews in Scotland demonstrated that similar effects could be generated using a low power violet diode source of only 300 µW intensity [23]. Diode laser sources are small, inexpensive, and relatively straightforward to implement, requiring a fraction of the footprint and infrastructure associated with more traditional lasing systems. As such, they provide a convenient route to poration in many routine processes, although at such wavelengths are intrin-sically limited to short working distances and cells in monolayer culture.

2.2.2.1 Near-Infrared porationWhile short wavelengths provide a direct route to membrane poration, lipid mole-cules are essentially transparent to these wavelengths meaning that almost all photon absorption is provided by the proteins and other biological macromolecules within the cell. The overall laser energies used are low, but it is important to remember that the actual beam flux within the focal volume is almost frightening in its intensity: these beams are typically focused to a diffraction-limited spot of around 500 nm radius. 300 µW in such an area is equivalent to ≈400 MW/m2: to provide a sense of scale to this vast number, this may be compared to the nominal solar irradiation at the Earth’s equatorial surface of ≈1000 W/m2. Such intensities inevitably cause a range of photochemical effects and damage among the tissues that the researcher is typically most interested in investigating, and this damage is not always immediately obvious. Membrane pores heal rapidly due to the fluidity of the lipid membrane,

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and the lipids are largely unaffected by such processes. By contrast, vast numbers of free radicals are formed within the electron-rich proteins and nucleic acids which control and regulate cellular processes, causing crosslinking and photolysis both be-tween them and internally within their structures. As a result, cell viability is often severely compromised. Worse still, these short-wavelength photons are absorbed by the very first such structure they encounter: as the degradation at these wavelengths is typically a single photon process, damage can occur outside the target region, both above and below the focus in the sample. This means that such approaches are per-fectly suitable for examining cells in a confluent monolayer, provided a tight focus is achieved to minimize off-target effects, but are completely unhelpful when working with three dimensional structures which are much closer to biological reality.

In order to alleviate these issues, substantial efforts have been invested into the exploitation of two-photon absorbance phenomena (Chapter 2 discusses the basics of two-photon absorption for photopolymerization). This quantum mechanical effect allows two or more identical photons’ energies to be summed if they arrive at an ab-sorbing structure coincidentally: that is, they arrive at the target in phase and at exactly the same moment [24]. Two infrared photons of λ = 1064 nm may thus combine to contribute the effects of one of 532 nm, for example. This is of particular relevance as biological tissue is almost completely transparent to near-infrared wavelengths, providing a mechanism to image and manipulate cells through a significant depth of surrounding tissue. Near-infrared beams are capable of penetrating a depth of some millimeters through biological material, although an increasing amount of light is scat-tered as the depth increases; for imaging and poration experiments conducted across a range of tens or hundreds of microns, however, such losses can be negligible.

Using a coherent source such as a laser ensures that the photons will arrive at an atom or delocalized molecule-sized target in phase, but even with bright laser sources, the odds of them arriving at exactly the same time become vanishingly small. However, the negligible absorbance of the photons through normal processes at these longer wavelengths means that enormous intensities from ultrashort laser pulses may be used with little effect on the surrounding tissue. Effective use of the technique comes when the incident beam is focused with a high numerical aperture lens to a narrow volume and, in particular, a short depth of focus: this squeezes the light into a confined space and maximizes the local flux, essentially stacking the odds such that many of these improbable events occur through simple weight of numbers. The two-photon absorption efficiency increases with the square of intensity, meaning that outside of this narrow focal region the effect tends to zero, while inside it two-photon absorption may be quite efficient.

Initially identified and applied in laser-scanning microscopy, Tirlapur and König were the first to apply the technique to DNA transfection, using high-intensity fem-tosecond laser pulses [25]. Mohanty et al. expanded the approach for more general optoporation experiments [26]: using a single, nanosecond pulse from a Nd:YAG laser at its primary emission band of 1064 nm [22] and a dye known to show strong two-photon absorbance, they were able to demonstrate the uptake of a range of mem-brane-impermeable dyes and also showed successful transfection with GFP-encoding

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plasmids. Stracke et al. further explored the mechanistic of this technique, demon-strating the uptake of ≈500 kDa fluorescein-labelled dextran molecules with a nomi-nal diameter of 13.3 nm [27]. More recently, the work of Torres-Mapa et al. has shown the uptake of these dextran molecules, roughly equivalent in size to an oligonucle-otide of 760 base pairs, by live Pomatoceros lamarckii embryos utilizing an all-optical workstation [28]. This system is based on a Ti:sapphire laser that was switched be-tween pulsed and continuous-wave modes, providing a contact-free system capable of manipulating and porating cells with a single laser. A practical guide to the technique has been published by the same group, providing an extremely useful resource for those wishing to explore the applications within their research [29].

While these techniques allow poration through a depth of unaffected tissue, it is important to note that the same photochemical effects that blight short-wavelength techniques are also an issue within the focal volume here. Two-photon techniques change the way in which the energy is delivered and tightly defines the volume in which it is absorbed, but once absorbed that energy will have exactly the same effect as if delivered by a single, short-wavelength photon. Furthermore, the high infrared intensities required pose issues of their own. While their absorbance by biological tis-sue is minimal, it is nonzero: the extinction coefficient of water at 1064 nm is around 10−1 cm−1 (see Jacques for a comprehensive analysis of light absorption by different tissues) [30]. As a result, the high flux required by some methods introduces heat-ing effects which may induce artefacts of their own. This is not an effect limited to two-photon approaches—the same issues arise in optical trapping processes, for ex-ample—but two photon techniques generally require higher intensities and it is easy to neglect the influence of the infrared beam when interpreting data. Liu et al. found that Chinese Hamster Ovary cells experienced a rise in temperature of 1.15 ± 0.25°C when exposed to a 100 mW diffraction-limited trapping beam of 1064 nm wave-length [31]. While such a rise is often insignificant, this may be sufficient to induce a range of aberrant behavior when applied to cells which are already destabilized by the formation of pores or the administration of other stress-causing agents.

2.2.3 Delivery/capture efficiencyWhile the formation of membrane pores or endocytotic invaginations is an essential part of the transfection and/or delivery process, it is not in itself sufficient for the process to take place. Pores formed in cell membranes are relatively passive struc-tures: upon opening, spontaneous exchange of material will take place with the sur-rounding medium, driven by diffusion gradients and electrostatic effects. This leads to a large degree of uncertainty in the doses of payload received by the target cell, particularly in instances when large, slowly-diffusing molecules are to be delivered. Longer poration events lead to greater volumes of material exchanged, but there is a finite amount of leakage and uptake that a cell can tolerate before sustaining irre-versible damage, and if artefacts are to be avoided then this period should ideally be minimized. As such, it is often convenient to bundle payloads into discrete parcels which may then be manipulated to the site of poration using light robotic techniques. Polyethylenimine (PEI) reagents are a simple way to deliver nucleic acids [32]: the

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long-chain polymers feature a high cationic charge density at physiological pH, meaning they readily bind to the electron-rich nucleic acid backbone and collapse to form “polyplexes.” Commercially available systems are often tailored to form particulate polyplexes of around 1 µm diameter, which are amenable to trapping and manipulation in their own right. PEI polyplexes and related cationic macromolecular structures will trigger endocytosis and transfection, and prevent lysosomal digestion of their payloads through their extremely high capacity for protons [33]. By coupling these with a poration technique, the process can be dramatically accelerated and provides essentially quantitative transfection efficiency. This system was used in our own laboratories to precisely deliver GFP-encoding plasmids to targets chosen from a range of human-derived cell lines [34]. However, the persistent nature of the polymers means that doubts persist about their medium- and long-term cytotoxicity, leading to the development of a range of alternatives including an elegant solution from Drake et al. which utilizes reversible disulfide bridges to form analogous struc-tures from biodegradable, small-molecule precursors [35].

A similar, solid-phase approach is also useful when attempting to abstract mate-rial. Rather than have the analytes of interest escape into the growth medium, greatly diluting their concentration even in the smallest of microfluidic structures [36], it is often more practical to collect them with an optically-steered microtool. This can be accomplished using surface coatings or chemistries chosen to maximize interac-tions with the target of choice: in our own research, we have achieved some notable successes using cationic lipid mixtures coated around polystyrene or silica cores to harvest cell membrane proteins [37]. However, a range of more specific recogni-tion moieties, such as antibodies or DNA oligomers may be bound to micro- and nanoparticle carriers, which have been critically reviewed elsewhere [38].

3 SECONDARY TARGET TECHNIQUESIn order to minimize the effects of optical absorption upon the tissues of interest, one technique is to avoid the target tissue altogether and focus the activating la-ser beam onto some other reactive surface or structure nearby. These surfaces can transduce the supplied energy into a number of forms through either laser induced breakdown and plasma formation or through straightforward thermal conversion (as described earlier in Section 2.1.1). This has the useful advantage of eliminating pho-ton damage among the cells and organelles under investigation, while the use of nano- or microparticle targets means that the hotspots generated are intrinsically lo-calized, providing a high degree of precision if the particles are trapped or otherwise immobilized.

However, the approach also brings a range of potential issues regarding acute and chronic cytotoxicity: nanoparticles can be taken up by even intact cells, leading to a range of atypical behaviors depending on their size and surface chemistry [39,40]. Some films and nanoparticles may also leach toxins from their core over time, lead-ing to long-term toxicity even when the cells’ short-term behavior is unchanged: for

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example, this effect underpins the in vitro toxicity of the vast majority of quantum dots, which otherwise appear to offer near-ideal properties for biochemical investi-gations. The applicability of this toxicology data in vivo remains a matter for debate [41], but for the purposes of investigative cellular and molecular biology, the tools are of limited value in long-term studies. Microparticles and films are not normally absorbed by cells with intact membranes, but the nature of many poration techniques means that beads may be taken up while pores are open, or encapsulated along with intended payloads during endocytosis events. Films can become degraded over time or repeated irradiation, leading to the generation of fragments which act as jagged-edged particles with a distribution of sizes. While the particles themselves are often biologically inert, their sheer size relative to the cell may introduce problems as they are introduced to the cluttered and crowded cell interior—particularly if repeated deliveries are attempted, as most are not biodegradable.

Many nano- and microparticles may also have important impacts on the mem-branes of the cells they encounter. The capping reagents used to coat the surface of many such particles to enhance their colloidal stability or toxicological behav-ior are often amphiphilic or surface active in nature. This is generally an intrinsic property—after all, their job is to act at the interface between a solid particle or oil droplet and its aqueous environment—but the same property that makes them useful also means they preferentially adsorb onto or intercalate between the lipid structures exposed on a cells’ surface. Sometimes this leads to the particles capturing a surface coating (called the “protein corona,” hypothesized to be the trigger for nanoparticle endocytosis) [39] and continuing on their path through the system: when carefully tuned, this can form the basis of an effective tool for cell surface biopsy [42]. In many cases, however, what is observed is the essentially irreversible incorporation of the bead or particle into the membrane structure, and the end of the experiment. Experimental issues aside, with careful material choice and fabrication both particles and films can provide powerful tools for the manipulation of cell membranes.

3.1 GOLDGold films and nanoparticles offer a simple, flexible, and low-toxicity target for the manipulation of cell membranes and related material, and are probably the most widely-used optical targets in use today. While a relatively expensive reagent, gold’s inertness makes it a very versatile choice of substrate in biological systems, while the strong (but noncovalent) bonds it forms with thiol compounds mean it can be read-ily functionalized with bespoke surface coatings. Gold can also be electroplated or otherwise deposited onto other materials as a thin film [43], masking their reactivity and allowing the production of multifunctional nanomaterials, such as the “spheri-cal nucleic acids” described by Cutler et al. [44,45], which couple surface-mounted DNA oligomers for protein detection with an inorganic core to facilitate capture and readout, separated and linked by a film of gold (shown schematically in Fig. 10.4). Gold particles may also be prepared in a wide range of form factors, meaning they can be custom-synthesized to provide highly-efficient plasmonic heating in response

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to irradiation at specific wavelengths [46]. To avoid their aggregation, they are typi-cally coated with a hydrophobic monolayer to enhance their colloidal stability: care-ful modification of this “nonstick” coating provides a highly flexible substrate with which to work [47].

Exactly as in the case of other materials, (Section 2.1.1), gold particles or surfac-es can be driven to laser-induced breakdown by means of a high-intensity laser pulse, or can utilize plasmonic heating to trigger gentler effects using continuous-wave sources. Arita et al. in the laboratories of Kishan Dholakia in St Andrews were the first to use the laser-induced breakdown of gold nanoparticles for transfection [48]. Their experiments showed the energy required for the breakdown of gold nanopar-ticles was a 1000-fold lower than that required for the equivalent effect in water, promising a much gentler technique. However, the breakdown still results in the formation of a cavitation bubble, meaning that the technique suffers from many of the same drawbacks highlighted in Section 2.2.1. Lower power and/or shorter pulses will generate smaller bubbles, however. Interesting recent work by Boutopoulos

FIGURE 10.4

A schematic spherical nucleic acid, showing a nanometer-dimension core structure containing a payload or magnetic structure that may be used for steering purposes. This is encapsulated in a thin film of gold, which may be modified by utilizing the high affinity that sulfur atoms show for the metal. Targeting DNA or RNA oligomers (shown as spirals) may be coupled to these thiols with alkyl linkers (represented as cylinders) which combine to form a continuous monolayer on the surface, generating a spherical coating of nucleic acids which shield the core from interactions with their surroundings.

3013 Secondary target techniques

et al. suggests that under such conditions, it is clusters of gold nanoparticles which mediate membrane poration, rather than the response of individual beads [49]. This offers a novel and orthogonal method to control the process: optical or chemical switching of nanoparticle aggregation could provide a valuable new approach to im-prove spatial precision at the subcellular level.

Optical heating using continuous wave or lower-power pulsed sources offer a very different tool. By coating a gold nanoparticle or rod in a hydrophobic mono-layer as described earlier, its affinity for the core of a lipid membrane increases exponentially. This means that the particle concentration is preferentially enriched at exactly the location one might wish to target in order to trigger phase changes in the structure of such a membrane, changes that are typically accessible within a few kBT of physiological conditions in native membranes in order to facilitate processes like endocytosis [50]. This combination of temperature sensitivity and the ability to generate localized thermal gradients has been utilized by researchers in both syn-thetic membrane systems and in vitro experiments. Paasonen et al. were the first to demonstrate the effect’s utility, using 250 nm UV light on gold nanoparticle-loaded liposomes of synthetic lipid mixtures [51]. They were able to show light-triggered release of dye from their vesicles at a temperature that varied with the membranes’ composition, exactly as might be predicted in a phase-change process. Fong et al. added a significant layer of sophistication, developing near-IR-sensitive systems based on plasmon enhanced absorption that released their payload from a cubosome structure: one that forms insoluble, biocompatible aggregates that are much more suitable for the long-term, on-demand delivery of therapeutic compounds such as insulin [52]. The approach has also been used to successfully trigger membrane fu-sion between two distinct bodies for the delivery of encapsulated material and the generation of hybridomas: this is discussed extensively in Chapter 11.

3.1.1 Organelle specific targetingAt around the same period, McDougall et al. combined these two techniques to dem-onstrate the injection of single gold nanoparticles across a cell’s nuclear membrane [53]. In these experiments, an optically-trapped nanoparticle was injected using a millisecond-duration dose of pulsed 780 nm laser light. This was sufficient to gener-ate a membrane pore through which the particle could travel, but insufficient to cause its collapse and subsequent cavitation, demonstrating the ability to inject individual, nanoscale payloads into specific volumes or organelles within live cells. While the throughput of these techniques is very low in this embodiment, it is clear how such a technique might be coupled to flow-based systems in order to deliver a truly practi-cal, light robotics-based platform for single cell stimulation or genetic modification.

Careful customization, however, allows nanoparticle uptake to be controlled at both the cellular and organelle level. A full review of the surface chemical and struc-tural modifications identified for such purposes is beyond the remit of this chapter, but the interested reader is directed to the recent, comprehensive work of Yameen et al. [54]. Once in position, gold nanoparticles are excellent probes for subcel-lular investigations: not only are they useful as heating and poration targets, but

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they permit characterization of their immediate environment in live cells through techniques such as surface-enhanced Raman spectroscopy. Huefner et al. have dem-onstrated the ability of such functionalized nanoparticles to distinguish between closely-related phenotypes of human neuroblastoma-derived cells [55], and it is easy to see how such research might be combined with controlled poration processes to either study or destroy selected cells within a population.

3.1.2 ToxicityWhile gold is not toxic at a bulk scale (hence its use in a variety of medical applica-tions, such as false teeth), there is conflicting information regarding its properties as a nanomaterial. Debate around the topic is fierce and reflects uncertainty around the potential for intrinsic toxicity of nanomaterials due to their size and morphology as much as their accumulation or reactivity. In depth discussion of the arguments are beyond the scope of this chapter, but the interested reader is directed to a recently-published summary by Fratoddi et al. [56].

A neat technique to sidestep the problem has been presented by Kalies and co-workers, who have immobilized gold nanoparticles onto the cell culture substrate us-ing a silanization reagent [57]. This technique incorporates the nanoparticles onto the surface of a thin film of biocompatible polydimethylsiloxane that is formed in situ in the culture chamber. This places the nanoparticles in direct contact with cells grown in the test vessel, but prevents them from being absorbed during poration experi-ments. Such surface functionalization experiments are explored in more detail later, but provide a simple route to delivering single-cell resolution to a broad population of target cells while minimizing or eliminating the potential toxic effects that such reagents might present if added as suspended particles.

3.2 THIN FILM DEPOSITIONIf the desired effects are accessible thermally, membrane manipulation can be made accessible to a much broader range of laser wavelengths and powers through the use of optically absorbing thin films. A range of films and structures have been uti-lized to controllably disrupt cell membranes: Yamane et al. have demonstrated that microdiscs of biocompatible titanium film, selectively patterned onto a coating of polydimethylsiloxane (PDMS), can be excited by a nanosecond laser to generate cavitation bubbles with a lifespan of a few 100 ns [58]. These cavitation bubbles can be utilized to disrupt membranes in much the same way as the plasma described earlier in Section 2.2.1, but with substantially lower magnitude forces involved. This technique has some disadvantages relative to those in Section 3.1.2 as the micro-discs are relatively large in size (≈2 µm) and sparsely-packed compared to the gold particles. This generates relatively widely-spaced active regions within the culture chamber and restricts the experimenter’s precision and choice of targets, although multiple discs may still be deployed within a single well. While this technique still fundamentally relies upon the biology traveling to the apparatus, it provides at least some degree of subcellular selectivity.

3033 Secondary target techniques

The use of amorphous silicon has also been well-developed and characterized. Its ubiquitous use in the production of solar panels means its optical behavior is well-understood, and it may be grown into atomically flat surfaces using well-established and widely-available plasma-assisted chemical vapor deposition techniques [59], although there remains some controversy about the mechanism(s) of its formation, recently summarized and contributed to by Kuwahara et al. [60] Mechanistic details notwithstanding, the properties that make it the material of choice in photovoltaic ap-plications also confer substantial benefits in its use as a substrate for light robotics. It is an optically-accessible semiconductor with a band gap of around 1100 nm, mean-ing that it absorbs weakly in the infrared while strongly absorbing photons of higher energies, appearing a deep red when held to the light. As in a photovoltaic system, the absorbed photons promote electrons into the material’s conduction band; how-ever, when not incorporated into a doped structure, the electrons and holes rapidly recombine leading to high localized heating. The amorphous, nanocrystalline struc-ture conducts heat very poorly [61], helping to limit the lateral spread of transient, laser-mediated hotspots and maintaining high thermal gradients, which are useful in providing spatial control in light robotic applications. More useful still, amorphous silicon is completely biocompatible and readily metabolized, meaning that cells can be cultured directly onto surfaces so treated, or can coexist healthily with silicon microparticles suspended in their medium.

In our own laboratories, we have recently utilized this material to demonstrate subcellular poration and transfection of specific, adherent cells of interest within their native culture [34]. This offers substantial advantages over many of the mi-crofluidic techniques described in Section 2.2.1.1, as trypsinization of the targets is not required and there are negligible off-target effects among neighboring cells. The pores generated were demonstrated to have dimensions of only a few nanometers in diameter, measured using quantum dot exclusion experiments, and healed within a timescale of seconds after the cessation of illumination. These effects combined to provide quantitative transfection efficiencies and survival rates, as well as providing a novel light robotics tool that is extremely amenable to automation and incorpora-tion into single cell studies.

3.2.1 Bubble oscillationAn alternative use of this technique is to generate a rapidly oscillating vapor bubble at some point removed from the cells of interest, and to use the pulsed pressure waves so generated as a tool to trigger mechanical membrane disruption in a man-ner analogous to the cavitation experiments outlined earlier in Section 2.2.1 [62,63]. By avoiding the catastrophic collapse of a cavitation bubble, much gentler pressure waves are produced which may be controlled precisely by varying the intensity and frequency of the pulsed laser used to trigger its formation. As previously, the ob-served effects are closely linked to the proximity of the target cell(s) to the oscillating microbubble but the gentler forces involved mean that a far greater degree of control is available than in more conventional plasma-bubble experiments. Furthermore, the use of a thin film of amorphous silicon as an optothermal substrate means that longer

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wavelengths of light may be used, minimizing or eliminating its absorbance by the cells within the vessel and, likewise, the impact of any photochemical effects that might be observed with higher energy photons.

The generation of oscillating bubbles cause by microsecond laser pulses induces a thermocapillary flow in the surrounding medium. Streaming caused by the bub-ble’s oscillations have been visualized and quantified by Ohta et al. using a range of microparticles to reveal a toroidal region of effect with a radius of around 30 µm from the bubble’s locus and a z-axis range of ± 12 µm, although this is obviously a function of the applied laser intensity [63]. These bubbles can be combined into arrays to generate quite complex fluidic systems: for example, the same group have generated an array of 12 individually-addressable microbubbles for more complex pumping operations [64], based on a holographic trapping system derived from the Red Tweezers spatial light modulator control software of Bowman et al. [21]. The transient, optically mediated micropumps formed through this process are among the most immediately accessible and applicable light robotics tools developed to date.

3.2.2 Optoelectronic toolsAnother application of thin-film amorphous silicon lies in the exploitation of its photoconductivity. As a semiconductor, silicon is highly resistive until a percent-age of its electrons are promoted into its conduction band through the absorbance of photons or some other applied field. In practice, this means that a sheet of the material can be made to conduct at specific points by shining light spots onto it; the short mean free path of these electrons once outside the area of illumination is such that conductance decays almost immediately beyond the edges of any illuminated regions. The pseudoterminals so created can then be used for techniques such as electroporation, providing effectively diffraction-limited electrodes that can trigger poration or lysis within a specific cell or region of cell.

This has been neatly demonstrated by Witte and coworkers, who proved that in-dividual cells of interest could be lysed without interfering with their neighbors when illuminated in a microfluidic electroporation cell coated with amorphous silicon [65]. Perhaps more interestingly, the same group have demonstrated unusual selectivity in their technique: the process was demonstrated to selectively lyse red over white blood cells [66], presenting a tool with which a blood sample might be selectively enriched to simplify the isolation of rare species, such as circulating tumor cells.

A major advantage of this approach is that the equipment necessary to implement it is remarkably simple. Optoelectronic methods utilize the light only as a mechanism to induce conduction in the thin film of amorphous silicon, meaning that the intensi-ties required are vanishingly small relative to the laser techniques described earlier. As a result, illumination can be provided from a traditional incandescent bulb or LED; such light-shaping as is required can be effectively produced using pinholes in a sheet or card, or using a PowerPoint slide and a regular projector, as demonstrated by Witte and coworkers [65]. Furthermore, the electrical gradients required are ex-tremely accessible: while gradients in excess of 10,000 V/m are typically required to induce electroporation effects in cells [67], these optoelectronic techniques utilize

3053 Secondary target techniques

microfluidic chambers of only around 100 µm in internal height, meaning that the total applied voltage is measured in single figures. While a range of complex mi-crofluidic fabrication processes are available to provide advanced chip geometries, the processes work equally well in a rudimentary chip constructed of two sheets of indium-tin oxide-coated glass and a well fashioned from double-sided tape, making the technique an excellent starting point for proof-of-concept studies.

3.2.3 Microparticle targetingThin film materials are extremely useful targets for optically-mediated membrane targeting, but bring with them a range of complications. The chemical vapor deposi-tion techniques typically used for their formation require temperatures of several 100˚C [68], and are thus incompatible with the vast majority of polymer species, particularly those used in the ultraprecise two-photon polymerization that is such a cornerstone of light robotics tool manufacture [69]. While recent advances have identified low-temperature techniques by which thin-layer films may be so deposited, (most notably electron beam physical vapor deposition) [70], these polymers are not the only targets available for these techniques and it is becoming apparent that a wide range of optically absorbing structures may be effectively used to mediate membrane responses. Similar membrane disruption effects have been demonstrated using mi-cro- or nanoparticles of a number of diverse materials: structures whose small size and large, tunable surface area offer a range of opportunities that may eventually lead to the application of light robotics tools in vivo or even in clinical medicine. Analysis of the literature also suggests these are not new phenomena: that indirect optical po-ration of cell membranes has been observed a number of times in studies, but that in very few such cases have the mechanisms or potential applications been rigorously explored. Their potential value is such that the identification and refinement of these tools must become a major focus of research over the coming decade.

A number of multifunction optically controlled microtools have been developed in recent years, (most notably from the laboratories of Jesper Glückstad in Copen-hagen), using two-photon fabrication techniques which stem from the technology described in Section 2.2.2.1 [71]. However, few if any have been described which can directly and controllably influence the cells with which they are in contact. Car-bon nanoparticles have been demonstrated to porate cells in a manner similar to that observed using thin layer amorphous silicon [72]: while the mechanism postulated by the authors is somewhat speculative and substantial cell mortality was observed, cells were demonstrated to take up a range of dyes and plasmids through dynamic, nanoscale channels. In this instance, the selectivity and switchability were provided by spatial control of laser irradiation, given the nanoparticles’ relatively uniform distribution throughout biological tissue. However, the authors were also able to demonstrate orthogonal particle steering, by doping their nanoparticles with 1%–2% iron during their synthesis. This makes the particles magnetic, meaning that they can be steered to desired regions through the application of strong electromagnets. The combination of the two techniques, such as using magnetic fields to direct particles to a desired region followed by high-precision illumination of single cells of interest,

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is an innovative take on a field that is typically dominated by optical trapping mo-dalities. Another such optical approach, discussed in Chapter 7 by Hayakawa et al. combines the carbon-based photothermal functionality with a polymeric structure to form a “hybrid nanorobot” capable of selectively porating a target cell [73].

The reason these techniques are so valuable is that they offer a number of adapta-tions and a new, orthogonal degree of precision to light robotics: particles may be optically trapped and manipulated to specific cells of interest or even regions within those cells, then activated using a laser pulse of a different frequency to trigger mem-brane poration or lysis of only those cells in their close proximity. It is this switch-ability that is their most potent promise: the ability to selectively trigger biochemical processes independently of the applied trapping beam.

Possibly the closest example to the ideal of a switchable system was elegantly dem-onstrated by Waleed et al. [74] Their experiments utilized a continuous-wave trapping beam and coaxially-mounted femtosecond pulsed poration laser: using this arrange-ment, they were able to trap a plasmid-coated microparticle and use it as a gunsight for the poration laser to target a specific region of cell membrane. This provided exquisite control over the transfection of individual cells within the bulk: both microparticle and pulsed laser were required in order for any new gene expression to be observed. This technique has obvious drawbacks when operating in a confined and three-dimensional biological environment, but demonstrates a novel all-optical system capable of deliver-ing a payload to an individual cell and inserting it through the membrane.

3.2.3.1 Effects of topography on nanoparticlesThe vast majority of nanoparticles are spherical, formed by vapor condensation or solution-phase aggregation. However, exploiting crystal-growth or etching tech-niques can lead to a range of alternative structures and topographies which may offer novel properties. Nanowires or nanorods are such examples: they can be formed through metal-assisted etching processes using reagents such as hydrogen fluoride to form nanostructured needles of a variety of diameters and lengths. These nanowires offer a range of unusual optical properties, including polarization sensitivity and modified plasmonic resonances, but can be further customized to provide exquisite control of conditions in the surrounding microenvironment. The techniques were pioneered by Pauzauskie et al., whose work in 2006 demonstrated that a range of diverse, high-aspect-ratio nanowires could be selectively trapped and manipulated, as well as effectively welded into structures using focused laser beams [75].

There is some discussion about the mechanisms of trapping such structures: met-al nanoparticles experience a significant scattering radiation pressure which ordinar-ily prevents their stable trapping, but those made of gold and many semiconductor materials may be effectively manipulated, suggesting some form of plasmonic inter-action [76,77]. Such interactions may also contribute to the divergent heating effects observed by other researchers in Pauzauskie’s team, who have recently demonstrated that substantial increases in photothermal heating are observed when point defects are incorporated into such nanowire structures [78]. This flexibility and broad toler-ance for customization make nanowires ideal for a wide range of photonic systems,

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from waveguides to biophysical probes. Chapter 8 discusses the optimization of nanowires for waveguiding and harmonic generation. A full account of their usage is beyond the purposes of this chapter, but a comprehensive discussion of the topic may be found in the work of Guo and coauthors [79].

4 CONCLUSIONSA wide and expanding range of light robotics tools exist for the extremely precise manipulation and poration of individual cell membranes, even among the crowded and chaotic conditions found in cell culture and tissue samples. The precision offered by light robotic and photonic techniques is such that not only can individual cells be targeted, but individual organelles within those cells may also be selectively studied or disrupted.

Light robotics offers an ideal tool for biological manipulations, primarily because it can be applied in a contact-free, sterile environment. For those systems requiring the cleanest conditions, the direct interactions of light with the target cells may be utilized to trigger a range of destructive or nondestructive cell membrane responses. However, many experiments are better suited to the lower-power irradiance required to trigger responses using secondary targets, an approach which helps to minimize the off-target damage suffered through photochemical processes or area-of-effect damage. These secondary targets must be introduced to the system of study and may pose some risks in terms of both acute and chronic toxicity, but the flexibility they provide to the experimenter is unparalleled. A range of probes and nanostructured tools can be synthesized with precisely-known and -controlled properties, tailored precisely to the demands of the system. These may be steered or activated through a number of orthogonally-triggered processes, providing precisely-calibrated power to only those materials of interest and minimizing off-target effects. While the de-velopment of these techniques is still the focus of active research, we are rapidly approaching an era of the all-optical biochemical workbench, providing all the tools of the traditional biochemical laboratory but at the single or subcellular scale and in a contact-free environment.

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313

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00011-2Copyright © 2017 Elsevier Ltd. All rights reserved.

Azra Bahadori, Lene B. Oddershede, Poul M. BendixNiels Bohr Institute, University of Copenhagen, Copenhagen, Denmark

CHAPTER OUTLINE

1 Introduction ...................................................................................................... 3141.1 Background and Applications ..........................................................3141.2 Molecular Fusion Machinery ...........................................................3151.3 Energy Barriers for Fusion ...............................................................3161.4 Optically Controlled Fusion .............................................................317

2 Optical Control of Plasmonic Nanoheaters .......................................................... 3192.1 Experimental Details ......................................................................3192.2 Optical Confinement of Metallic Nanoparticles ..................................3192.3 Temperature Near an Irradiated Nanoparticle ....................................3222.4 Quantification of Nanoscale Heating on Membranes ..........................322

3 Fusion of Pure Membranes ................................................................................ 3263.1 Controlled Vesicle-Vesicle Fusion .....................................................3273.2 Verifying Fusion by Mixing of Lipids and Vesicle Lumens ...................328

4 Fusion of Pure Membrane Systems to Living Cells ............................................... 3304.1 Controlled Cell-Vesicle Fusion .........................................................3314.2 Verifying Lipid and Lumen Mixing in Cell-Vesicle Fusion ....................3314.3 Cell Viability After Cell-Vesicle Fusion ..............................................332

5 Fusion of Two Selected Living Cells ................................................................... 3345.1 Controlling Cell-Cell Fusion .............................................................3345.2 Verifying Cell-Cell Fusion by Lipid and Lumen Mixing ........................3345.3 Viability After Cell-Cell Fusion .........................................................3365.4 Optimizing the Fusion Process ........................................................337

6 Conclusion and Outlook ..................................................................................... 338Acknowledgments .................................................................................................... 338References .............................................................................................................. 338

Optically controlled fusion of selected cells and vesicles using plasmonic nanoheaters

11

314 CHAPTER 11 Optically controlled fusion of selected cells

1 INTRODUCTION1.1 BACKGROUND AND APPLICATIONSBy gaining control over biological processes one can learn more about the mecha-nisms governing life and potentially utilize this knowledge to progress the devel-opment of biorobotics. Bioinspired robotics can be used to study and manipulate biological systems, and to solve bioengineering problems in an automated manner. With the development of high quality, easily controllable, miniaturized, and low-cost lasers, light is an excellent tool for controlling biological matter. The current chapter deals with the usage of optical manipulation to perform an optically controlled fusion of two selected entities. These two entities can be anything that is surrounded by a membrane, for instance vesicles or living cells.

Membrane fusion is a ubiquitous phenomenon in biology facilitating a range of important processes like intracellular fusions involved in synaptic transmission, fer-tilization, or fusion mediated entry of enveloped viruses [1–5]. These processes are essential to life and therefore, from an engineering perspective, it is highly desirable to control fusion of biological entities, preferably in a nontouch, noninvasive, and automated manner. Considering the many compartments within cells it is not sur-prising that cells have developed highly specialized fusion complexes to carry out a variety of fusion processes to allow merging of separate compartments. For instance, transport by exo- or endocytosis in or out of the cell is efficiently performed by fusion or fission of small lipid vesicles as schematically depicted in Fig. 11.1 [2] and also intracellular transport from the Golgi network to the plasma membrane relies on fission and fusion of membranes. Another example is intercellular communication in the synaptic cleft, which relies on fusion of intracellular vesicles to the plasma membrane and consequent content delivery into the intercellular gap [6]. Cells also employ other types of morphological membrane structures in intercellular commu-nication; for instance, elongated membrane protrusion are found in many cell types, these can emanate from one cell and contact and fuse to other receptor cells. Such structures are initially called filopodia [7–10], but evolve into tunneling nanotubes [11], which are efficient and stable tracks for specific transport between cells.

In addition to being essential to the homoeostasis of cells, membrane fusion also offers exciting possibilities for manipulating and controlling cells at a molecular level as well as for creating interesting hybrid structures through fusion of two cells, also called a syncytium [12].

Optical control of selective cell-cell fusion and the construction of hybrid cells have significant translational impact for basic research in drug delivery and for cel-lular design. Optical control of fusion also has important applications in cellular research. Cells fused from different cell types can, for example, be used in hybridoma technology for production of monoclonal antibodies [13], the result of fusion of stem cells with differentiated cells can be used for diabetes treatments through pancreatic islet transplantation [14], and fusion of dendritic cells to a triply negative breast can-cer has been shown to be a promising new tool for developing new vaccines [15].

3151 Introduction

This chapter presents a new optics based strategy utilizing nanoscale plasmonic heating to induce membrane fusion in a selective and controlled fashion. Membrane properties are extremely temperature dependent and membranes locally change properties if exposed to localized heating. If two membranes are in close contact and are heated at the contact zone by an irradiated metallic nanoparticle, the two mem-branes undergo rapid fusion. In this chapter we present the experimental details of the method and show examples of fusion between (1) two vesicles (Section 3), (2) a vesicle and a living cell (Section 4), and (3) between two living cells (Section 5).

1.2 MOLECULAR FUSION MACHINERYInspired by the abundance of fusion events observed in biology, researchers have put effort into reproducing or mimicking the fusion strategies that cells employ. To overcome the barrier for merging two membranes it is critical to bring the two mem-branes into close proximity and subsequently destabilize the bilayer structure thereby

FIGURE 11.1 Examples of Membrane Fusion Events Occurring Inside Cells

A vast number of different proteins, depicted as small symbols, facilitate different and highly specialized fusions ranging from mitochondrial fusion and vesicular fusion to the plasma membrane. Some of the proteins important for fusion are SNARE complexes (filled red circles) and viral fusion proteins (pink triangles).

Adapted with permission from Martens S, McMahon HT. Mechanisms of membrane fusion: disparate players

and common principles. Nat Rev Mol Cell Biol 2008;9:543–556 [2].

316 CHAPTER 11 Optically controlled fusion of selected cells

allowing the two membranes to merge into one single bilayer. There are several ways to bring membranes into close contact and thereby increase the likelihood of fusion. Molecular fusion machineries exist in nature like, for example, the SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) complex which is a set of proteins specialized in bringing membranes together in a zippering fashion to induce fusion [5]. To better understand the mechanism behind such fusion complexes synthetic fusion complexes have been developed which closely resemble the func-tion of the naturally occurring SNARE proteins. These fusion complexes include single stranded DNA with complementary strands anchored to lipids on apposing membranes [16–18]. Membranes that get into nanoscale proximity by random diffu-sion will be forced even closer together by the hybridization of these complementary DNA strands in a zippering fashion, analogously to the zippering of SNARE proteins.

1.3 ENERGY BARRIERS FOR FUSIONThe many different types of fusion processes in biology ranging from viral fusion, cell-cell fusion to intracellular vesicle fusion are mediated by different types of proteins. These proteins facilitate fusion by delivering the energy required to bring the mem-branes together and merge the bilayers. The steps in membrane fusion are depicted in Fig. 11.2. The first step in Fig. 11.2A involves forcing the bilayers together to establish a contact area with separation distances within the nanometer or subnanometer range. This step requires water to be removed from the space between the membranes. Since the water layer closest to the membrane is bound to the hydrophilic lipid heads, it does require a significant amount of energy to completely dehydrate the intermembrane space, and this energy can be supplied by SNARE proteins as presented schematically in Fig. 11.2F. Subsequently, the membrane bilayer has to form a fusion pore, which can occur through highly localized bending of the bilayers as shown in Fig. 11.2B,C followed by merging of the proximal leaflets of the two membranes into a hemifu-sion state (Fig. 11.2D). Finally, the hemifused membrane can transform into a single closed membrane by formation of a fusion pore (Fig. 11.2E).

The energy density (energy per area) associated with the local bending of a mem-brane with principal and local radii of curvature R1 and R2 can be expressed in terms of the local principal membrane curvatures C1 = 1/R1 and C2 = 1/R2 as well as the local Gaussian curvature which is given as the product of the principal local curva-tures Cg = C1 × C2 [20]

κ κ= + − +E C C C C C( /2)( )b 1 2 02

G 1 2 (11.1)

where kb and kG are the bending and Gaussian moduli of the membrane, respectively, and C0 is the spontaneous curvature of the membrane. The bending modulus of a cell membrane is typically on the order of 10–100 KBT and hence spontaneous bending into high curvature regions is unlikely.

The subsequent steps in fusion are depicted in Fig. 11.2 and involve merging of the proximal leaflets (called hemifusion) and finally formation and expansion of a fusion pore which allows the contents of the two compartments to mix.

E=(kb/2)(C1+C2−C0)2+kGC1C2

3171 Introduction

1.4 OPTICALLY CONTROLLED FUSIONThe existence of an energy barrier preventing spontaneous fusion has triggered development of several techniques, which can induce membrane fusion. By perform-ing controlled fusion one can study the energetics of fusion [21,22], create hybrid cells [12], and perform drug delivery to cells [23]. Most techniques used for trigger-ing membrane fusion are able to either expand or open the bilayer to form a fusion pore, thus minimizing the barrier for fusion. For creation of a fusion pore energy

FIGURE 11.2 Steps in the Fusion of Lipid Bilayers

(A) Establishment of a contact area between two membranes. (B) Inducing local curvature minimizes the energy of the hydration repulsion between the proximal leaflets of the membranes. (C) Formation of a fusion stalk with only proximal leaflets fused whereas the distal leaflets are not fused. (D) A hemifusion diaphragm forms as a result of stalk expansion. (E) Formation of a fusion pore and subsequent expansion of the pore allows the contents of the two compartments to mix. (F) Steps involved in protein mediated fusion of a synaptic vesicle to a flat membrane mediated by SNAREs. (i) Complementary and cognate proteins from the SNARE protein family, depicted as red and blue, (ii) form hybrids and bring the two membranes into close contact in a zippering fashion. (iii) A fusion intermediate forms with fusion of the proximal membrane leaflets (iv), followed by fusion of the distal leaflets, and (v,vi) consequent opening of the fusion pore and transfer of the content across the membrane.

Part A–E: Adapted with permission from Ref. Chernomordik LV, Kozlov MM. Mechanics of membrane fusion.

Nat Struct Mol Biol 2008;15;675–683 [4]; Part F: Adapted with permission from Boal DH. Mechanics of the

cell, 2nd ed. Cambridge University Press: Cambridge; New York, 2012 [19].

318 CHAPTER 11 Optically controlled fusion of selected cells

has to be supplied to expand the bilayer, through tension [22] or by generation of high local curvature [24] which can expose the hydrophobic interior of the bilayer and catalyze fusion. As membranes are highly responsive to electric fields, fusogenic pores can be generated in both fluid and gel phase membranes [25] by applying electroporation to giant unilamellar vesicles (GUVs) whereby fusion can be induced between GUV membranes [21] or cells [15] that are in close contact.

Another technique for triggering fusion involves disruption of the cell membrane and thereby triggering fusion. This can be done by using either high energy pulsed lasers [26] or ultraviolet lasers [27,28] which can easily ablate biological material. Various methods for cell membrane disruption are discussed in Chapter 10. Inducing fusion alone by continuous wave lasers requires relatively high powers of ∼100 mW at the focal region (corresponding to ∼10 MW/cm2). Fusion typically uses highly focused laser beams and, consequently, the divergent light cone illuminates a large zone below and above the focal point where the actual fusion occurs, thus resulting in light exposure of a large part of the sample. Fusion mediated by pulsed lasers and UV lasers is carried out at laser wavelengths which interact strongly with the biological material and such approaches typically compromise the viability of cells. Another drawback related to both pulsed UV lasers and electroporation is the inability to select the cells for fusion, or to manipulate the location of the fusing cells or vesicles which then become arranged randomly and need to be positioned by microfluidic approaches or by other means.

Near infrared (NIR) continuous wave (CW) light, on the other hand, is well suited for optical trapping as it is much less harmful to biological material due to low absorption [29]. Consequently, fusion cannot be achieved by NIR lasers alone using this wavelength regime. However, by combining highly focused NIR laser light with absorptive plasmonic nanoparticles, it is possible to deposit a substantial thermal energy at a nanoscale spot (plasmonic heating) at the contact area between mem-branes and fusion can be triggered due to the increase in local temperature. Like ten-sion and curvature, which are frequently used regulators for a number of processes in biology including endo- and exocytosis [24,30,31], temperature also has significant impact on the state of membranes. Exposing fluid membranes to increasing tempera-tures leads to bilayer expansion of 0.5%/°C, whereas a temperature change across the phase transition temperature of a membrane leads to ∼20%–25% change in area of the bilayer [32,33]. Cell membranes are much more complex than simple compo-sitions of a few lipid species, but they have been shown also to respond to changes in temperatures and exhibit critical temperatures near the physiological temperature [34,35]. Hence, high local temperatures can lead to disruption or expansion of the bilayer with consequent exposure of the hydrophobic core to water thereby creating highly fusogenic regions which can fuse to nearby membranes.

High local temperatures can be obtained by laser irradiating metallic particles which absorb light by interaction with surface plasmons [36]. The heating of irradi-ated nanoparticles has been quantified in a relevant bilayer assay where the local temperature increase has been found to easily reach 200–300°C and extend only few tens of nanometers from the nanoparticle surface [37]. The fact that the zone with

3192 Optical control of plasmonic nanoheaters

increased temperature is highly spatially confined together with the fact that the near infrared laser light (λ = 1064 nm), causes minimal biological photodamage [38], makes this technique particularly promising for fusion of membranes and cells while still ensuring cell viability.

With the development of advanced types of automated optical trapping of both metallic nanoparticles and larger biological specimens it becomes a natural strategy to combine plasmonic heating with optical trapping to provide a toolbox to selec-tively fuse individual cells. This will pave the way for more automated future appli-cations by using light for high throughput and controllable fusion of cells.

2 OPTICAL CONTROL OF PLASMONIC NANOHEATERS2.1 EXPERIMENTAL DETAILSImaging and trapping was carried out on a platform consisting of a Leica SP5 con-focal microscope and an optical trap based on a 1064 nm laser (Spectra Physics J201-BL-106C), details on the equipment are given in Ref. [39]. The output powers were 200–750 mW, at the sample plane, assuming an objective transmission of 60%. Both the trapping and imaging lasers were focused by a Leica PL APO, NA = 1.2, 63X water immersion objective. The strength of the optical trap was improved by adjusting the collar of the microscope objective to minimize spherical aberration [40]. Since the optical trap was stationary, the relative movement of trapped versus untrapped cells/GUVs was facilitated by a piezoelectric stage (PI 731.20, Physik Instrumente, Germany) which allows lateral movement of the sample with respect to the optical trap with nanometer precision. To detect the gold nanoparticles (GNPs), we exploited the strong light scattering of the metallic particles. By collecting the backscattered light, in the spectral range 465–483 nm, from trapped gold nanoparti-cles irradiated by a 476 nm argon laser line, the gold nanoparticles could be detected by a photomultiplier tube.

Cells were suspended in a glass bottom Petri-dish together with GUVs and AuNPs. The dish was mounted on the microscope stage where the sample tempera-ture was kept at 37°C during the experiment. Vibrant@DiO, FAST-DiO, calcein, and calcein AM fluorophores were excited using a 488 nm laser line, and emitted light was collected in the spectral range 500–577 nm. Vibrant@DiD was excited using a 633 nm laser line and the emitted intensity was collected in the spectral range 640–759 nm.

2.2 OPTICAL CONFINEMENT OF METALLIC NANOPARTICLESMetallic nanoparticles placed in an optical field interact strongly with light [41–43]. Conduction electrons confined by the small size of the particle oscillate with a characteristic frequency called the surface plasmon frequency at which the particle exhibits particularly pronounced absorption and scattering of light [42,43]. Metals have a finite penetration depth for light, quantified by the skin depth, which depends

320 CHAPTER 11 Optically controlled fusion of selected cells

on the wavelength of the incident light. If the nanoparticle is small compared to the skin depth, it becomes approximately uniformly penetrated by the light and the nanoparticle-light interaction can be described as a dipole interacting with a light field. Dipoles are sensitive to the gradient of the light intensity and become effec-tively attracted toward the highest intensity [42]. This has been exploited in single beam optical trapping of several types of metallic nanoparticles made of gold or silver [42,44–47].

The optical force on a nanoparticle can be split into two contributions, namely (1) the radiation pressure arising from scattering plus absorption and (2) the gradient force.

The force arising from the radiation scattering and absorption is given by:

=FPn

cCrad ext

(11.2)

where n is the index of refraction, c is the speed of light, and <P> is the time aver-age of the Poynting vector which denotes the directional flux of energy density. Cext is the extinction cross section which equals the sum of the absorption cross section, Cabs, and the scattering cross section, Cscat.

The gradient force is given by:

α= ′ ∇F1

2| | 2E (11.3)

where α′ is the real part of the polarizability of the particle and <E2> denotes the time

average of the squared electric field and is proportional to the light intensity. From Eq. (11.3), it is clear that the gradient force points along the direction of the intensity gradient, that is, toward the highest intensity which is at the focus of a laser beam. Metallic nanoparticles can be readily trapped at an off resonance condition where the trapping wavelength is much longer than the surface plasmon resonance wavelength [42]. We note, however, that as the particle size increases there is a significant red-shift in the surface plasmon resonance frequency and consequently both the absorp-tion, and in particular the scattering cross sections, increase significantly as shown in Fig. 11.3E,F. The large scattering cross section complicates stable trapping of larger metallic nanoparticles which are several hundreds of nanometers in diameter.

The particle size and shape might vary considerably within the same batch of gold nanoparticles, as shown in Fig. 11.3A–D, where the displayed nanoparticles were claimed to be spherical by the manufacturer. Since shape and size critically defines the absorption and scattering characteristics, as shown in Fig. 11.3E–F, the heating produced by the individual irradiated nanoparticles might vary significantly within one batch depending on the exact characteristics of the individual nanoparticle.

The extinction cross section is given by [43]

α π= + = + ″C C C k ka| | /4ext scat abs4 2 (11.4)

where α″ is the imaginary part of the complex polarizability, α, and k is the wave-number, k = 2πn/λ. Since α scales with volume (for small particles compared to the

Frad=nPcCext

F=12|α|∇E2

Cext=Cscat+Cabs=k4|a|2/4π+ka''

3212 Optical control of plasmonic nanoheaters

skin depth) [43], we see from Eq. (11.4) that the absorption, and, consequently, the particle temperature, will scale with the volume of the particle. Similarly, the scatter-ing term scales with α2 or with the square of the volume which leads to destabiliza-tion of the optical trap when the particle size becomes too large.

When designing applications where plasmonic heating is desired together with optical trapping it is important to maximize the heating, but still ensure that one retains the ability to efficiently optically trap the particles. Therefore, interme-diate sizes which correspond to diameters around 100 nm, are often a good choice since heating and optical trapping efficiency can both be increased by increasing laser power and the ratio between scattering and absorption is not too high, hence minimizing the risk that the particle is pushed out of the trap.

Quantification of the trapping strength can be achieved by performing a stan-dard calibration of the optical trapping potential. For small particles and small dis-placements, the trapping potential is harmonic if the laser intensity has a Gaussian

FIGURE 11.3 Characterization of Gold Nanoparticles Used in the Optically Induced Membrane Fusion Presented in This Chapter

(A–D) Transmission electron micrographs (TEM) of citrate stabilized gold nanoparticles with nominal diameters of (A) 80 nm, (B) 100 nm, (C) 150 nm, and (D) 80 nm (streptavidin coated). Scale bars are 100 nm. (E) Measured absorbance for the particles shown in (A–C). (F) Calculated and normalized extinction cross-sections for spherical gold nanoparticles ranging in size from 10 to 125 nm in radius. The vertical dashed line denotes λ = 1064 nm, a laser wavelength frequently used in optical trapping applications.

The particles shown in (A–C) were purchased from British Biocell International (BBI) and the particles in (D)

were purchased from NANOPARTZ.

322 CHAPTER 11 Optically controlled fusion of selected cells

intensity distribution in all directions. However, the exact strength of the trap is not important when using the trap to facilitate membrane fusion between vesicles or cells, the only important issue is that the trap should be capable of trapping the nanoparticle at the contact zone between the vesicles or cells and should be able to position the vesicles or cells next to each other. Therefore, we do not explain the cali-bration procedures here, but instead direct interested readers to details in Chapter 1 and literature describing calibration of optical traps [48–50].

Trapping of the particle should be done at as low laser powers as possible while still achieving fusion of the membranes. This is important to achieve efficient fusion while avoiding excessive heating that may harm the living cells or disrupt the mem-brane systems.

2.3 TEMPERATURE NEAR AN IRRADIATED NANOPARTICLELocal heating of micron sized regions by laser irradiation can be applied for manipu-lating the motile behavior of cells, as discussed in Chapter 13, or to make polar blebs in cells [51]. Irradiation of an absorptive nanoparticle results in more efficient and more localized conversion of light energy to heat [36]. Due to the small size of the nanoparticle we can regard the emission of heat as coming from a point source which makes it possible to analytically describe the symmetric temperature distribution as a function of distance to the nanoparticle. The steady state temperature increase, ∆T, as a function of distance, D, to the center of a nanoscopic heat source can be written as [52],

∆ =T DCI

D( ) (11.5)

where I is the light intensity and C is a constant which contains several physical parameters like the thermal conductivity of the environment, neglecting the thermal conductivity of the membrane which is only a few nanometers thick. If we con-sider a nanoparticle with radius R, then the temperature increase at the surface of the particle is given as: ∆T(R) = CI/R. For a given particle size and laser intensity the temperature increase therefore simply decays as one over distance to the heat source, 1/D.

2.4 QUANTIFICATION OF NANOSCALE HEATING ON MEMBRANESSeveral strategies have been developed to measure the temperature of single irradi-ated nanoparticles, however most of the strategies fall in one of the three following general categories:

1. Detection of temperature-dependent physical changes of the surrounding medium, for instance, changes in the index of refraction or viscosity [53–57].

2. Detection of temperature dependent changes in fluorescence intensity or polarization near the irradiated nanoparticle [58,59].

∆T(D)=CID

3232 Optical control of plasmonic nanoheaters

3. Detection of a thermal phase transition of the medium surrounding the irradiated nanoparticle either in a 2- or 3-dimensional matrix in which the irradiated nanoparticle is embedded [37,42,60–62].

Examples of these approaches are detailed in the respective references. Chapter 13 uses approach (2), where some experimental details and calibration data are avail-able. Here we detail how approach (3) can be used to detect the temperature of any irradiated nanoparticle which generates sufficient heat to melt surrounding lipid bilayers or lipid membranes. This approach is highly relevant in the context of con-trolled optical induction of membrane fusion since the membrane itself is used as a temperature sensor in this method.

Briefly, to determine the temperature profile the irradiated nanoparticles are placed on a gel phase membrane. An area of the membrane around the irradiated particle will melt if the temperature increases above a critical melting temperature as shown in Fig. 11.4. By using phase sensitive fluorophores, for instance fluorophores that prefer to partition into the melted phase, the melted zone around the nanoparticle can be visu-alized as shown in Fig. 11.4B. At the rim of the melted zone, the temperature equals the critical melting temperature of the lipid (for instance Tm = 33.8°C for the lipid used in [63]). In the temperature-distance relationship between a nanoheater and its envi-ronment [Eq. (11.5)], C can be found because the distance (Dm) to rim of the melted region where T = Tm, is known. When C is known it is possible to quantify the entire temperature profile around the irradiated particle including the surface temperature of the nanoparticle. The temperature of the irradiated nanoparticle increases with its size as shown in Fig. 11.4C–D. Particles with diameters of 80 nm were found to reach a temperature increase of ∼50°C for laser powers used in successful fusion of pure membranes, P ∼ 200 mW at the focal plane [64], whereas 150 nm gold nanoparticles reach temperature increases exceeding 200°C for laser powers used in fusion of cells where P ∼ 350 mW at the focal plane. Such temperatures are more than sufficient to ablate biological material and thereby locally disrupt membranes. The photothermal heating of several types of metallic nanoparticles with different shapes and composi-tions have been quantified using a membrane as a temperature sensor [37,61,63,65].

While the assay presented in Fig. 11.4 is well suited for quantifying particle tem-peratures, it cannot fully elucidate the effect of an optically trapped metallic nanopar-ticle on membranes in three dimensional settings where the particle is free to diffuse in the optical potential, however, for this purpose a similar 3D assay can instead be employed as demonstrated in [61].

Optical trapping of plasmonic nanoparticles near membranes can change the membrane permeability and shape as the temperature reaches the phase transition temperature. By using GUVs made of the same type of lipids as was used in Fig. 11.4 (DC15PC), it was shown that trapping gold nanoparticles (diameter 80 nm) near the GUV (Fig. 11.5A) resulted in a local temperature increase (Fig. 11.5B) and conse-quently in significant permeability changes of the membrane concomitantly with the onset of the phase transition, Fig. 11.5C–D [61,66,67]. The onset of a phase transi-tion was also backed up by an increase in the bilayer hydration as measured with

324 CHAPTER 11 Optically controlled fusion of selected cells

FIGURE 11.4 Membrane Based Assay for Quantifying the Heating From an Irradiated Nanoparticle

(A) Irradiating a nanoparticle placed on a two-dimensional lipid bilayer (DC15PC) results in melting of the nearby membrane in a region where the temperature is higher than the phase transition temperature of the lipid bilayer. The temperature increase around the irradiated particle scales inversely with the distance D from the nanoparticle and at the edge of the melted region the temperature equals the phase transition temperature Tm of the lipid bilayer (DC15PC). Experimental detection of the distance from the particle to the rim of the melted zone thus allows quantification of the particle temperature. (B) Experimental imaging of the melted zone by using a fluorescent dye (FAST-DiO, ThermoFisher Scientific) which selectively partitions into the fluid part of the bilayer. A 150 nm GNP positioned at the center of the melted footprint (yellow overlay) is irradiated using a λ = 1064 nm laser at P = 10.3 MW/cm2. Scale bar is 2 µm. (C) Binary images of experimental data showing how the melted region increases with increasing laser power. Each color corresponds to a given laser power. The three images (from top to bottom) correspond to 200, 100, and 80 nm gold nanoparticles irradiated using an optical trap based on a 1064 nm laser. Scale bar is 8 µm. The depicted gold nanoparticles are not drawn to scale with the melted areas. (D) Surface temperature increase as a function of laser power for particles with different diameters: blue asterisks 80 nm, black squares 100 nm, yellow diamonds 150 nm, and red circles 200 nm. Dashed lines are theoretical predictions based on Mie theory.Part A,D: Adapted with permission from ACS Nano 2010;4:2256–2262. Copyright (2010) American Chemical

Society; Part C: Image adapted with permission from Andersen T, Kyrsting A, Bendix PM. Local and transient

permeation events are associated with local melting of giant liposomes. Soft Matter 2014;10:4268–4274 [65].

3252 Optical control of plasmonic nanoheaters

the environmentally sensitive fluorophore Laurdan or di-4-ANEPPDHQ, Fig. 11.5C [67]. Heating is known to result in deformation [67,68] and expansion [32,33] of membranes and this can also be detected if a GUV is heated sufficiently fast by an optically trapped nanoparticle [67].

Interestingly, it has been found that gold nanoparticles moving in an optical trap-ping potential achieve very different temperatures than gold nanoparticles which are irradiated while immobilized on a surface [37,61]. Apart from the different thermal conductivity of the environment in the two setups (glass–water interface versus only water), this is explained by the fact that the tightly focused optical trap exhibits a significant degree of spherical aberration with a complex three-dimensional intensity distribution [69,70]. Some particle sizes were found to be trapped at local inten-sity maxima with a much lower intensity than at the focus volume, thus explaining the lower observed temperature [69,70]. One can therefore consider the temperature measured with the two-dimensional assay in Fig. 11.4 as an upper estimate for the heating of a similar particle free to move inside the potential of an optical trap. This

FIGURE 11.5 Optical Trapping of a Gold Nanoparticle (80 nm) Near a GUV Made From DC15PC (Tm = 33.8°C)

(A) Schematic of a gold nanoparticle optically trapped in a focused laser beam near a GUV loaded with self-quenched calcein. (B) A surface plot of a typical temperature profile. (C) Example of a GUV undergoing a local phase transition. The bright region corresponds to the melted area where the dye (di-4-ANEPPDHQ) up concentrates as it has a preference for the fluid phase [66]. The dashed line indicates the distance at which the temperature corresponds to the phase transition temperature of the lipid bilayer. Scale bare is 2 µm. (D) The GUV contains calcein at self-quenched concentrations (green color) and upon leakage, induced by the phase transition, a fluorescent burst is detected near the permeation site due to dilution and consequently dequenching of the fluorophores. The red color on the GUV membrane comes from the overlay with the membrane which is labeled with TR-DHPE. Scale bare is 10 µm.

Part C,D: Images reproduced with permission from Andersen T, Bahadori A, Ott D, Kyrsting A,

Reihani SN, Bendix PM. Nanoscale phase behavior on flat and curved membranes. Nanotechnology

2014;25:505101 [66].

326 CHAPTER 11 Optically controlled fusion of selected cells

is in particular the case when using an optically trapped gold nanoparticle to mediate fusion, since the particle will most likely be pushed away from the center of the trap by the nearby membranes.

3 FUSION OF PURE MEMBRANESThe phrase “pure membranes” in this chapter represents the membrane of GUVs (diameter: 10–200 µm), which are constructed by the standard electroformation method from a hydrated lipid film with a known composition.

The word vesicle denotes a spherical, self-assembled lipid bilayer system of microscopic dimensions which consist of one or more bilayers of natural and/or synthetic lipids and entrap an aqueous volume [71,72]. Vesicles with only one bilayer membrane are known as unilamellar vesicles. According to their size, unilamellar vesicles can be subdivided into three main classes, namely, (1) small unilamellar lipid vesicles (SUVs, d ≤ 100 nm), (2) large unilamellar lipid vesicles (LUVs, 100 nm < d ≤ 1 µm), and (3) giant unilamellar lipid vesicles (GUVs, d > 1 µm), Fig. 11.6A [73].

Fig. 11.6B shows a confocal image of typical fluorescently labeled (TR-DHPE, 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-Texas Red) electroformed GUVs, which are made of the phospholipids DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPS (1,2-dioleoyl-sn-glycero-3-phosphoserine) purchased from Avanti Polar Lipids. Phospholipids are the major constituent of natural mem-branes [75]. They are known as amphiphilic molecules due to the presence of a highly polar head group and two hydrophobic tails on the same molecule [71,75]. When amphiphiles are dispersed in an aqueous solvent, the hydrophobic-hydrophilic interactions drive them to self-assemble into structures whose geometry is deter-mined by the competition between the free energy and the entropy of the system [20].

Depending on the effective shape of the amphiphiles and the extrinsic thermody-namic conditions, phospholipids can self-assemble into different structures [76,77]. The bilayer sheet is one such structure. It is a two-dimensional structure where the polar moiety of each of the building blocks (i.e., the cylindrical phospholipids) faces the polar environment (water phase) whereas the hydrophobic tails of the phospho-lipid are shielded from the polar solvent (Fig. 11.6A). Formation of a curved vesicle involves two competing energy contributions; the energy associated with having a flat membrane sheet with hydrophobic edges exposed to water and the energy asso-ciated with bending a flat bilayer into a vesicle [20,78]. The energy associated with having free edges exposed to water is high and membranes typically self-organize into closed spherical shapes.

Electroformation is an efficient method for forming GUVs and relies on applying external AC electric fields on a lipid film while the film is being hydrated on a conduc-tive surface (e.g., an indium tin oxide coated glass surface). The external AC electric field interacts with the intermembrane charges thereby facilitating bilayer separation and bending which are two important prerequisites for vesicle formation [79].

3273 Fusion of pure membranes

3.1 CONTROLLED VESICLE-VESICLE FUSIONMembrane fusion has received considerable attention due to its biomedical impor-tance [13–15]. Being able to select two objects for fusion and to control the time for fusion is clearly beneficial in nano chemistry using controlled vesicle-vesicle fusion with pico- or femtoliter volumes of reactants.

In Ref. [64], a highly selective and generally applicable physical method for trig-gering membrane fusion was first reported. Optical tweezers were used to position two selected GUVs in close proximity. A controlled fusion of the two selected vesi-cles was triggered by using the optical tweezers to irradiate plasmonic nanoparticles located in the contact zone between the two membranes. The local plasmonic heating was shown to be sufficient to overcome the energy barriers of fusion pore formation. The consequent membrane expansion [32,33] caused a complete fusion between the two selected GUVs, where both bilayers and the two cargos completely fused [64]. This strategy of first trapping two GUVs and bringing these in close contact provides full control over which vesicles are going to undergo fusion. Hence, light robotics in the form of tightly focused laser beams, optical traps, are highly useful for control-ling and mediating fusion. Over the last decades, optical tweezers based on a single tightly focused Gaussian laser beam have been shown as a powerful tool to remotely displace microscopic objects whose refractive index is higher than their surrounding medium. Although vesicles are only made of a thin membrane (≈5 nm for unilamel-lar vesicles), it has been shown that they can also be optically trapped and displaced in 3D if their lumen is filled with a solution with a sufficiently higher refractive index

FIGURE 11.6 Classification of Unilamellar Vesicles

(A) Based on their sizes, unilamellar vesicles are classified into three main groups: small unilamellar vesicles (SUVs) which are less than 100 nm in diameter, large unilamellar vesicles (LUVs) with a diameter between 100 and 1000 nm; and giant unilamellar vesicles (GUVs) with a diameter larger than 1 µm. (B) Fluid phase GUVs made of DOPC and DOPS and labeled with fluorophores, TR-DHPE (red) or FAST-DiO (green). The scale bar is 10 µm.Part A: Reproduced with permission from Ribeiro MM, Melo MN, Serrano ID, Santos NC, Castanho MA. Drug-

lipid interaction evaluation: why a 19th century solution? Trends Pharmacol Sci 2010;31:449–454 [74].

328 CHAPTER 11 Optically controlled fusion of selected cells

compared to their surrounding buffer [80]. High concentrations of sucrose have been successfully used to make even SUVs (with diameters down to 50 nm in diameter) trappable by optical tweezers [80]. Therefore, optical tweezers and light robotics can play a critical role in the hot nanoparticle mediated membrane fusion: first, they are used to bring any pair of sucrose loaded GUVs of interest into close contact. Second, they are used to trap and irradiate metallic nanoparticles in the contact zone to pro-duce local plasmonic heating. As a result, membrane fusion is triggered in a highly selective and controlled manner.

3.2 VERIFYING FUSION BY MIXING OF LIPIDS AND VESICLE LUMENSAs a proof of complete GUV-GUV fusion, one can verify both the lipid and lumen mixing of two GUVs by suitable fluorescent labeling, as schematically demonstrated in Fig. 11.7A,D. In Ref. [64], confocal microscopy is combined with optical tweezers to visualize the lipid and lumen tracers during the optically induced membrane fusion. An example of such an experiment is shown in Fig. 11.7B where the two fusing mem-branes are fluorescently labeled by two different lipophilic dyes. Confocal imaging of such fluorescently labeled membranes allows for visualization of fusion pore opening and the formation of the newly fused vesicle. Quantitatively, the migration of each of the two different fluorescent dyes within the new membrane area can be tracked in real-time, as shown in Fig. 11.7C. A rapid decrease in the emitted intensity per area is seen for both fluorophores. This decrease is initiated at the onset of fusion when each fluo-rophore type redistributes in the increased membrane area created by fusion. Experi-mental data indicate that the time scale of mixing for each of the lipophilic fluorophores scales linearly with the area of the other GUV [64]. A theoretical model quantitatively describing the lateral diffusion in a membrane formed as a spherical shell can be used to theoretically estimate the time scales for mixing of membrane bound fluorophores which are initially distributed in separate hemispheres of a closed shell. The theoreti-cally predicted times scales are consistent with the experimentally measured time scales and thus confirm lipid mixing by diffusion in a two-dimensional membrane [64].

The method is generally applicable for a wide range of vesicles including GUVs of 10–200 µm in diameter, negatively charged/uncharged GUVs, and also for GUVs in different physical states (gel phase and/or fluid phase GUVs [64]). The time scale for lipid mixing depends on the physical state and the size of GUVs. As expected, when one or both of the fusing vesicles are in the gel state (at temperatures lower than the phase transition temperature), the time scale of lipid mixing increases by one order of magnitude [64].

The right half of Fig. 11.7 demonstrates cargo mixing after fusion. A sketch of plasmonic heating triggered membrane fusion and the associated cargo mix-ing is depicted in Fig. 11.7D and the corresponding confocal images are shown in Fig. 11.7E. The plot in Fig. 11.7F represents the diffusion of the hydrophilic green fluorophore (calcein) within the fused volume.

The intensity of calcein becomes uniformly distributed within the volume of the fused GUV within ∼1s as shown in Fig. 11.7F. The time scale for achieving complete mixing depends on the sizes of the individual GUVs and the size of the

3293 Fusion of pure membranes

diffusing molecule. The diffusion coefficient of a small water soluble fluorophore like calcein has been measured to be 333 ± 117 µm2/s [81] and therefore the lumens of GUVs with radii of ∼10 µm should undergo complete mixing on a time scale of ∼1s which is consistent with the data shown in Fig. 11.7F.

As the hot GNP mediated GUV-GUV fusion method is capable of inducing com-plete fusion including cargo mixing, it can be utilized to monitor interactions of the cargos after membrane fusion. As a biologically relevant application, protein-mediated membrane shaping has been triggered by this fusion method [64]. Two

FIGURE 11.7 Lipid Mixing and Cargo Mixing Associated with Hot Nanoparticle Mediated GUV-GUV Fusion

(A, D) Schematic representations of fusion between two adjoining GUVs by means of laser-induced heating of GNPs in the contact zone between the two GUVs. Fusion takes place in three main steps including (1) GUV positioning, (2) GNP trapping, and (3) fusion with associated lipid mixing and lumen mixing. (B, E) Confocal images of events corresponding to the schematics in (A) and (D), respectively. A white arrow points out the position of the GNPs (d = 80 nm). The lipophilic tracers FAST-DiO (green) and TR-DHPE (red) are used to label the membranes in (B). (E) Membranes are labeled with TR-DHPE and one of the lumens is filled with calcein (green). The scale bar is 10 µm in each image series. Upon fusion, both membrane lipids and vesicle lumens mix. (C) The emitted intensities from FAST-DiO and TR-DHPE are normalized over the area and plotted as a function of time. A drastic decrease in both intensities occurs upon fusion due to dilution of a conserved number of fluorophores into the larger membrane area of the fused vesicle. (F) The intensity emitted from calcein in the red and the blue boxed regions, shown in (E), are normalized over the area and plotted as a function of time. Calcein diffuses from the calcein containing GUV into the GUV containing no dye and finally it uniformly distributes inside the volume of the fused GUV.

Reprinted with permission from Bahadori A, Lund AR, Semsey S, Oddershede LB, Bendix PM.

Controlled cellular fusion using optically trapped Plasmonic Nano-Heaters Proceedings of SPIE.

2016;9922:1-10. Copyright (2016) Society of Photo Optical Instrumentation Engineers.

330 CHAPTER 11 Optically controlled fusion of selected cells

examples of such experiments are shown in Fig. 11.8A–B where a neutrally charged GUV containing I-BAR domains in the lumen is fused to an empty GUV composed of acidic lipids. I-BAR domains are conserved domains which are involved in mem-brane curvature sensing and curvature induction. The ability of the protein to shape and sense membrane curvatures is tightly linked to the convex-shape of its dimeric structure which binds preferentially to negatively charged membranes through the presence of a cluster of basic amino acids. Since the protein binds efficiently only to acidic membranes, membrane tubulation is only observed when the neutral GUV containing I-BAR domain is fused to a GUV containing negatively charged lipids, which activates the tubulation activity of the I-BAR domain.

4 FUSION OF PURE MEMBRANE SYSTEMS TO LIVING CELLSThe fusion method presented in the previous section for fusing GUV membranes is also applicable to living cells, which are surrounded by a plasma membrane with physical properties similar to the GUV membranes. Fluid membranes, also those

FIGURE 11.8 Fusion of I-BAR Containing GUVs Composed of Neutral Lipids with GUVs Composed of a Mixture of Acidic and Neutral Lipids Results in I-BAR Mediated Membrane Tubulation

(A) I-BAR domain (from the ABBA protein) is encapsulated in a FAST- DiO labeled (upper green) vesicle which is made of neutral DOPC. Images show the fusion of this GUV to a TR-DHPE labeled vesicle (lower red) which is made of 40% negatively charged DOPS lipids. Several distinct protruding tubes form after fusion. Scale bare, 10 µm. (B) YFP-tagged I-BAR (green color inside the upper GUV) is encapsulated inside a neutral DOPC GUV. The second, lower GUV, is composed of 30% negatively charged DOPS lipids. The membranes in both vesicles are labeled by DiD. In this experiment, a ∼10× higher concentration of ABBA I-BAR-protein results in a significantly larger number of formed tubes compared to that of panel (A) upon fusion. The YFP tagged I-BAR becomes diluted upon fusion and bleaches over time, thus it is not seen in the last image in panel (B). Scale bare, 10 µm.

The figure is adapted with permission from Nano Lett 2015;15:4183–4188.

Copyright (2015) American Chemical Society [64].

3314 Fusion of pure membrane systems to living cells

surrounding living cells, exhibit a relative expansion of ∼0.5%/K [33] and the local temperature elevation generated by a laser irradiated GNP can easily reach tens of degrees at the site of the nanoparticle. Thus, this local temperature increase is expected to significantly lower the energy barrier for forming a fusion pore in the membrane of a living cell and it might even locally disrupt the rigid actin cortex which supports the membrane in most cells.

An important issue related to cell fusion is the possible effect on the cell viabil-ity caused by the laser itself or by the plasmonic heating generated by the optically trapped GNPs. In the following section we describe hot nanoparticle mediated cell-GUV fusion and cell viability following fusion.

4.1 CONTROLLED CELL-VESICLE FUSIONFusing a vesicle to a live cell in a controlled manner allows for delivery of a cargo, for example, a certain drug or genes, to that particular cell. Using optical tweezers one chooses the cell and the sucrose loaded vesicle of interest and brings them into close contact. During movement of the GUV toward the cell it can happen that a gold nanoparticle diffuses into the trap, which could locally heat up the GUV, but this has no consequences since fusion can only occur when membranes are in close proximity. As for GUV-GUV fusion, cell-GUV fusion also occurs shortly, within seconds, after laser irradiation of GNPs in the contact zone of the two membranes when using the same laser power as for trapping the GUV. Fusion does not hap-pen by means of the laser alone, nor does it happen spontaneously if a GUV and a cell are brought into close contact. The presence of a hot nanoparticle in the con-tact zone is a necessary requirement for successful fusion. Even GNPs as small as 80 nm produce sufficient local heating to mediate membrane fusion between a live HEK293 cell and a GUV. However, cell-GUV fusion is more delicate compared to GUV-GUV fusion because care should be taken to minimize the possible heat induced damages to the cell integrity and therefore, the temperature should be kept as low as possible.

4.2 VERIFYING LIPID AND LUMEN MIXING IN CELL-VESICLE FUSIONTwo examples of cell-GUV fusion mediated by plasmonic heating are shown in Fig. 11.9. The fusion process is visualized by confocal microscopy. A time sequence of confocal images for each experiment is shown in Fig. 11.9A and C. In both cases, the GUV membrane is labeled with FAST-DiO (green) and the cell membrane is labeled with vybrant DiD (red). Fusion associated lipid mixing for the experiment displayed in panel (A) is verified by the time evolution of intensity per pixel emit-ted by the membrane fluorophores vybrant DiD (red) and FAST-DiO (green) in Fig. 11.9B. Upon fusion, each of the membrane tracers diffuse into a larger mem-brane area leading to a significant decrease in intensity at the time of fusion. The time scale of lipid mixing is found to be one order of magnitude slower for cell-GUV fusion (50 s) compared to fusion of two fluid phase GUVs (5 s) [64].

332 CHAPTER 11 Optically controlled fusion of selected cells

To verify content mixing after hot GNP mediated cell-GUV fusion, the cell cyto-plasm in the experiment shown in Fig. 11.9C is labeled with calcein AM. Calcein AM is invisible in the media, but it turns green upon enzymatic conversion to calcein inside the cell cytoplasm of living cells. As seen in Fig. 11.9D, the content mixing is verified by plotting the time evolution of the normalized intensity of green calcein in the GUV lumen (light green) and in the cell cytoplasm (dark green) after fusion. The time scale of content mixing in cell-GUV experiment is found to be at least one order of magni-tude slower, ∼30 s, than that of GUV-GUV fusion. This could be due to crowding in the cytoplasm or differences in the viscosity of the cytoplasm compared to the viscos-ity of the sucrose solution within the GUV lumen [82,83].

4.3 CELL VIABILITY AFTER CELL-VESICLE FUSIONAn assay involving the fluorophore calcein AM was used to assess cell viability after the fusion event. Calcein AM remains fluorescently inactive until its acetoxymethyl

FIGURE 11.9 Lipid Mixing and Content Mixing Associated with Hot GNP Mediated Cell-GUV Fusion

(A) and (C) are confocal images of fusion of a FAST-DiO labeled GUV (green) with a vybrant DiD labeled HEK293 cell (red). Laser powers of P = 650 mW and P = 750 mW (at the sample) were used to irradiate a GNP (d = 80 nm) in (A) and (C), respectively. The white arrows point at the GNPs. The scale bars in (A,C) are 10 µm. (B) The emitted intensities of vybrant DiD and FAST-DiO are normalized per pixel and plotted as a function of time. Lipid mixing is verified by a decrease in the emitted intensities from each of the lipophilic fluorophores upon fusion. The intensity spike in the FAST-DiO signal (green curve), occurring at the onset of fusion, is crosstalk originating from GNP scattering. (C) The cell cytoplasm is labeled with green calcein. (D) Time evolution of average calcein fluorescent intensity for the experiment shown in (C). The light green trace is from within the GUV, the dark green trace is from within the cytoplasm. It takes approximately 30 s for calcein to be uniformly distributed within the fused cell-GUV structure.

Parts A and B are unpublished results. Parts C and D are reprinted with permission from

Bahadori A, Oddershede LB, Bendix PM. Hot-nanoparticle-mediated fusion of selected cells.

Nano Res. Copyright (2017) Springer Nature Singapore.

3334 Fusion of pure membrane systems to living cells

ester bound is hydrolyzed which converts it to membrane impermeable fluorescent calcein [84,85]. The retention of the hydrophilic and fluorescent calcein inside the cytoplasm only occurs in cells with intact membranes and, hence, a fluorescent cal-cein signal can be used as a marker for cell viability.

A cell viability experiment is shown in Fig. 11.10. A time series of confocal images is shown in Fig. 11.10A where the FAST-DiO labeled GUV membrane is seen as green and the vybrant DiD labeled HEK293 cell is seen as red. The GNPs are seen as yel-low spots in the contact zone. Laser irradiation of the GNPs triggers membrane fusion

FIGURE 11.10 Viability Investigations of a Cell-GUV Fused Structure

(A) Images showing the hot nanoparticle mediated fusion between a FAST-DiO labeled GUV (green) and a vybrant DiD labeled HEK293cell (red). The fusing cell and the GUV are shown inside the blue ellipsoid; laser power is P = 400 mW at the sample. An optically trapped d = 80 nm GNP was used to trigger fusion. Calcein AM (nonfluorescent) was added to the chamber 6 min after fusion. The last image in (A) shows calcein fluorescence in the fused structure indicating that the cell is viable. Also, the cytoplasm of the control cells situated below the blue ellipse turns green. The scale bar is 10 µm. (B) A semilogarithmic plot of calcein intensity emitted inside the lumen of the fused structure (dark green) and inside the cytoplasm of the nonfused control cell (light green) as a function of time for the experiments shown in panel (A). The spikes in intensity occurring between 145 and 180 s are due to an experimental artifact caused by repositioning the stage to keep cells in the field of view. Inset in (B) is a schematic representation of the viability experiment with calcein AM.

Reprinted with permission from Bahadori A, Oddershede LB, Bendix PM. Hot-nanoparticle-mediated

fusion of selected cells. Nano Res. Copyright (2017) Springer Nature Singapore.

334 CHAPTER 11 Optically controlled fusion of selected cells

through local plasmonic heating and subsequently the GUV gradually collapses into the cell structure. The last image in Fig. 11.10A shows the result of a cell viability assay performed 6 min after fusion. As can be seen, the fused structure and the control unfused cell situated below the fused structure both turn green upon injection of non-fluorescent calcein AM into the sample chamber. Hence, the fused structure is viable. However, both qualitatively (from Fig. 11.10A) and quantitatively (from Fig. 11.10B), it can be seen that the emitted intensity from calcein inside the fused cell, although clearly detectable, has a lower intensity than inside the control cell. This can be partly explained by dilution of the cytoplasm following fusion to a GUV. Hence, the fused structure can retain the fluorescent calcein, which is signature of a viable cell.

5 FUSION OF TWO SELECTED LIVING CELLSThe creation of novel hybrid cells is of considerable technical interest since it allows for the formation of cells with mixed genetic information from both the original cells [12]. Here we demonstrate that the hot GNP mediated fusion is capable of triggering complete fusion of two selected HEK293 cells which thus form a viable syncytium with an intact plasma membrane.

5.1 CONTROLLING CELL-CELL FUSIONAs shown in Fig. 11.11, to perform controlled cell-cell fusion, first the two cells of choice should be positioned next to each other using an optical trap. Thereafter, one or more GNPs could be optically trapped at the contact zone by using the same optical trap. Fusion does not spontaneously occur between the two cells which are brought into immediate contact, neither can it be mediated by the NIR laser alone. As with the GUV-GUV and cell-GUV fusions, it is the laser irradiation of GNPs in the physical contact zone between the cell’s membranes which triggers cell-cell fusion. This is shown in the schematics in Fig. 11.11A panel II. In the confocal images shown in Fig. 11.11B–C, one vybrant DiO labeled HEK293 cell (green) and one vybrant DiD labeled HEK293 cell (red) are first brought into contact by an opti-cal trap. The optical trap is then located in the contact zone to confine a GNP with d = 150 nm. A fusion pore opening is induced by the generated plasmonic heating.

5.2 VERIFYING CELL-CELL FUSION BY LIPID AND LUMEN MIXINGAn example of successful fusion of two cells is shown in Fig. 11.11B. Both the plasma membrane and the internal membranes of cells are labeled with lipophilic dyes, and cytoplasmic mixing after cell-cell fusion can be directly inferred from the mixing of the internally labeled membrane structures. Upon fusion, the two cells col-lapse into one spherical volume and the two cytoplasms progressively mix due to the lack of any cell wall barrier in between. Within 30 min, the cytoplasm of the fused syncytium appears yellow because the originally red and green labeled cytoplasmic

3355 Fusion of two selected living cells

membrane structures are now thoroughly mixed. The two nuclei of the newly formed syncytium appear as the dark area from which the membrane dyes are excluded.

Fusion associated lipid mixing corresponding to the experiment shown in Fig. 11.11C is tracked over time, normalized per pixel and plotted in Fig. 11.11D. The average fluorescence per area of both green and red membrane labels decreases as the cells’ plasma membranes mix and the fluorophores are distributed over a larger area.

Similar to cell-GUV lipid mixing, the time scale of lipid mixing during cell-cell fusion is an order of magnitude slower than that of GUV-GUV fusion. This can be explained by the high diversity of phospholipids in the cell membrane including high levels of cholesterol which makes the cell membrane closely packed. Furthermore, the presence of cortical cytoskeletal structures affiliated with the membrane may slow down the mobility of phospholipids [86].

FIGURE 11.11 Cell-Cell Fusion Induced by Optically Heated GNPs

(A) Schematic of the fusion process: (I) An optical trap is used to position a cell of interest into close proximity of a target cell, (II) Fusion pore formation and membrane mixing are induced by plasmonic heating of one or more GNPs (d = 150 nm) located at the contact zone between the cells, (III) Lumen mixing confirms the complete fusion of the two cells. Nuclei are drawn as black ellipsoids. (B), and (C) show confocal images of hot GNP mediated fusion between one vybrant DiD labeled HEK293 cell (red) and one vybrant DiO labeled HEK293 cell (green). Laser powers at the sample are P = 250 mW and P = 350 mW for (B) and (C), respectively. The GNP (d = 150 nm) is seen as a bright spot in the contact zone and white arrows are used to show the location of the GNP. Orange arrows denote the position of the nuclei, which appear dark by effectively excluding the dyes. The scale bars are 10 µm. Last image of (B): within ∼30 min the interior lipid structures are thoroughly mixed and the overlap of the fluorophores results in yellow emission. (D) Normalized emitted intensities from vybrant DiO and vybrant DiD corresponding to the experiment shown in panel (C) plotted as a function of time. The intensities emitted within the ROIs depicted in (C) are plotted. Both intensities decrease because a fixed number of fluorophores diffuse to a larger membrane area upon fusion.

Reprinted with permission from Bahadori A, Oddershede LB, Bendix PM. Hot-nanoparticle-mediated

fusion of selected cells. Nano Res. Copyright (2017) Springer Nature Singapore.

336 CHAPTER 11 Optically controlled fusion of selected cells

5.3 VIABILITY AFTER CELL-CELL FUSIONTo test the integrity of the plasma membrane calcein AM is added to the cell medium as depicted in Fig. 11.12A. The confocal images shown in Fig. 11.12B are from the same experiment as shown in Fig. 11.11B, however, with the addition of a later image. Two hours after fusion, calcein AM is injected into the sample. The resulting green flu-orescence from calcein inside the newly formed syncytium indicates that it is biologi-cally competent. The graph in Fig. 11.12C shows the time evolution of the fluorescent intensity emitted from cytoplasmic calcein for the experiment shown in Fig. 11.12B. Time zero indicates the time at which calcein AM is added to the chamber. Within a time scale of a few minutes calcein AM diffuses across the cell membrane and is con-verted to fluorescent calcein which remains within the viable syncytium.

In the experiment presented in Fig. 11.12B, the fused syncytium is surrounded by a dashed blue ellipsoid. Above this, an unfused “control” cell is visible. As expected, this control cell also turns green upon adding calcein AM to the chamber. The cytoplasmic intensity levels of fluorescent calcein in both the control cell and in the fused syncytium are similar. This shows that the fused syncytium is equally viable as the control cell and that the hot GNP fusion method inflicts minimal damage to living cells.

By monitoring a larger number of control cells within the same sample, it is clear that the control cells do not always express a similar level of calcein fluorescence.

FIGURE 11.12 Viability After Cell-Cell Fusion

(A) Schematic representation of the cell viability assay in which nonfluorescent calcein AM is enzymatically converted to fluorescent calcein (green) after diffusing across the cell membrane and reaching the cytoplasm. (B) Viability experiment with the same cells as shown in the fusion experiment in Fig. 11.11B. After fusion of the two cells, calcein AM was flushed into the sample and diffused across the cell membrane of the syncytium which is surrounded by the blue ellipse. The diameter of the single GNP is 150 nm, laser power is P = 250 mW at the sample. The scale bar is 10 µm. (C) Cytoplasmic calcein fluorescence intensity plotted as a function of time for the experiment presented in (B). Time zero corresponds to the time when calcein AM is added to the chamber which is ∼2 h after cell-cell fusion. Unpublished results.

Reprinted with permission from Bahadori A, Oddershede LB, Bendix PM. Hot-nanoparticle-mediated

fusion of selected cells. Nano Res. Copyright (2017) Springer Nature Singapore.

3375 Fusion of two selected living cells

This is probably because their cell cycles are not synchronized and enzymatic activ-ity varies with cell cycle.

5.4 OPTIMIZING THE FUSION PROCESSThe continuous wave NIR (1064 nm) laser of the optical tweezers is nearly non-invasive since biological material has a minimum absorption at this wavelength [29]. In addition to the laser wavelength, there are several other factors that need to be considered to minimize possible damage to live cells during hot GNP induced fusion. For instance, the size of the GNPs and the power of the irradiating laser both influence the amount of heat emitted from the irradiated gold nanostruc-ture and hence determines the absolute temperature reached and the extent of the heated region [37]. GNPs with diameters in the range of 80–200 nm have been shown to successfully mediate membrane fusion using laser powers in the range of ∼200–750 mW at the focal plane. However, the largest GNPs in this range heat the local regions substantially up to 5 µm from the GNP [37] and this could affect the viability of the fusing cells. For each chosen GNP, the laser power should be optimized such that fusion readily occurs within a time scale of minutes while minimizing the generated heating.

Another important issue is aggregation of GNPs. Irradiation of a GNP aggregate will produce an overall higher heat generation compared to the heat generated by irradiation of a single GNP with the same laser power. Formation of GNP aggre-gates can be minimized by lowering the concentration of the GNPs in the sample chamber and by using PEG coated GNPs. Cell shrinking, cell rupture, and bleb formation are typical signs of thermal cell damage that should be avoided. Hot GNP induced fusion of two selected cells can result in a viable syncytium if sufficient care is taken to optimize the mentioned parameters. However, in the case of cell-GUV fusion, the fused structure often shows a somewhat compromised viability. This could be due to the large volume of the GUV in comparison to the volume of the cell, which causes the delivery of a relatively large volume of sucrose solution from the GUV to the cell with consequent dilution of the cytoplasm. Therefore, using smaller vesicles containing a solution with more physiological salt composi-tion would minimize the perturbation of the cell following cell-vesicle fusion. We note that when using vesicles which are too small, they will position at the center of the trap and thereby displace the GNPs from the center, thus making fusion more difficult.

Formation of GUVs containing more physiological solutions has been achieved by modifying the protocol for formation of the GUVs [87–89]. However, for the fusion technique presented here, it is critical to keep the index of refraction of the internal GUV solution higher than the external solution to enable optical trapping of the vesicle [80]. Altogether these possible routes for optimizing the fusion assay need further exploration, but will lead to more efficient and gentle fusion of biologi-cal structures with a wider scope of applications.

338 CHAPTER 11 Optically controlled fusion of selected cells

6 CONCLUSION AND OUTLOOKPlasmonic heating induced by irradiating metallic nanoparticles can efficiently medi-ate fusion between membranes. The irradiated nanoparticles need to be located at the interface between the two membranes of interest to induce fusion. To obtain this configuration, one can use an optical trap to manipulate the cells or GUVs of interest into close proximity and thereafter trap a plasmonic nanoparticle in the contact zone. The high local temperature induced by the irradiated nanoparticle causes the mem-branes to locally expand and expose their hydrophobic cores, whereby the formation of a fusion pore is feasible. In this chapter, we demonstrated how to fuse selected HEK293 cells and/or membrane vesicles with simple compositions. We expect that the laser controlled fusion technique presented here can be applied to fusion of dis-parate cell types to produce heterokaryons, and it might even be possible to fuse the nuclear membranes to produce synkaryons [12]. Also, the technique could be applied to biophysical studies of membrane vesicles with complex compositions and to study protein-membrane interactions as exemplified in Ref. [64] with binding of I-BAR domain proteins to acidic membranes.

The strategy of using optics and nanoparticles to induce fusion is a nascent tech-nique which has plenty of potential for further optimization. Using infrared resonant nanoparticles like, for example, gold nanoshells together with a NIR laser would dramatically increase the light to heat conversion and hence lower laser powers and smaller nanoparticles could be used to mediate fusion of the cells or vesicles. More-over, multiple optical traps [90–92] have been developed which could be used to perform fusion between multiple objects simultaneously. Optical trapping of several cells in 3D for interactions studies has been realized with mouse embryonic stem cells [93] and such strategies pave the way toward using light robotics for performing automated fusion between selected cells in a microfluidic device.

ACKNOWLEDGMENTSThe authors acknowledge financial support from the Lundbeck Foundation, the Villum Kann Rasmussen Foundation grant number VKR022593, the Danish Council for Independent Re-search DFF—4181-00196, the Danish National Research Foundation grant number DNRF116 and the Novo Nordisk Foundation grant number NNF14OC0011361.

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CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00012-4Copyright © 2017 Elsevier Ltd. All rights reserved.

Mark R. Pollard*, Neil M. Kad***DFM A/S, K. Lyngby, Denmark; **University of Kent, Canterbury, Kent, United Kingdom

CHAPTER OUTLINE

1 Introduction ...................................................................................................... 3451.1 Measurements at the Single Molecule Level .....................................3461.2 Examples of Single Molecule Optical Trapping Experiments ...............348

2 Force Probe Design ........................................................................................... 3502.1 Probe Production Processes ............................................................3512.2 Probe Release and Loading into Solution..........................................353

3 Experimental Setup ........................................................................................... 3553.1 Optics and Laser for Trapping ..........................................................3553.2 Methods for Creating Multiple Traps ................................................3573.3 Position Measurement ....................................................................3583.4 Additional Components ..................................................................361

4 Force Probe Calibration ..................................................................................... 3624.1 Initial Calibration of Laser Position ..................................................3624.2 Calibration of Force Probe Position ..................................................3634.3 Trap Stiffness Measurement ............................................................3634.4 Examples of Test Forces Applied to the Probe ...................................366

5 Single Molecule Force Experiment ..................................................................... 3696 Future Directions ............................................................................................... 377References .............................................................................................................. 380

1 INTRODUCTIONSingle molecule approaches are used to study biochemical processes fundamental to the function of cells and tissues. The processes that lend themselves most easily to single molecule imaging-based experiments involve movement. Among many such process exemplars include muscle contraction and protein–DNA interactions. The

The application of optically trapped force probes in single molecule biology experiments

12

346 CHAPTER 12 Application of optically trapped force probes

latter contribute to repair and replication, which are the foundation of cell reproduc-tion and growth. This chapter will report how the unique capabilities of optical trap-ping, used to detect picoNewton and femtoNewton forces that arise over nanometer distances, have been further augmented by using manufactured probes that detect forces using precise protruding tip structures. We report the design of the probes, the instrumentation and methodologies needed for the successful application of force probes in single molecule experiments.

1.1 MEASUREMENTS AT THE SINGLE MOLECULE LEVELBiochemical processes result from a vast number of interactions between molecules. These interactions are fundamentally stochastic in nature but organized as a system. Small defects in function can lead to systemic malfunction resulting in disease. The development of treatments relies on understanding these processes, which are often obscured by the average behavior of the ensemble, highlighting the importance of using single molecule methods. Therefore breaking down the incredibly complex nature of these processes is required. Simplified versions of the processes are created in vitro (from the Latin term, “in glass”) [1], outside of the living body but within controlled liquid environments. It is in these environments where optically trapped force probes are commonly applied.

The energies involved in single molecule experiments can be related to the aver-age thermal energy per particle in one direction, as described by the Equipartition theorem [2], where kBT is equal to 4 × 10−21 J at a temperature of 20°C. This energy can be related to the integral of force over a distance. A force transducer, such as an optically trapped probe, offers the means to measure this energy, and this value is equivalent to 4 pN nm. Therefore we need a force sensor capable of measuring picoNewton forces (or less) over nanometer distances. The fundamental principle for measuring forces with optically trapped objects is that they obey Hooke’s law, as shown in Eq. (12.1):

=F kx (12.1)

Where the force imparted on the probe (F) results in a displacement of x from the trap center, and k represents the trap stiffness. The optical trapping effect was first discovered by Ashkin [3] and the optical force that restores a trapped object to the trap center was found to be linear for a particular range of displacements of the object from the trap center, for example, where a 1 µm diameter sphere has a linear force response for displacements of up to 200 nm from the trap center [4]. This concept was utilized by Ghislain and coworkers to scan the surface of a manu-factured polymer layer [5], then applied by Florin and coworkers to detect picoNe-wton forces using an instrument called a Photonic Force Microscope [6], where spherical particles were trapped in order to scan the surface of biological cells. Subsequent development of the optical trapping technique has allowed smaller bio-logical samples to be studied, such as single molecules, which we will describe in the next section.

F=kx

3471 Introduction

A variety of force measurement techniques have emerged to investigate single molecule forces, the most commonly used being atomic force microscopy (AFM), magnetic trapping (MT), and optical trapping (OT). The forces that can be detected by these techniques and examples in biochemistry are plotted in Fig. 12.1.

Atomic force microscopy permits the widest range of forces to be measured, from nanoNewtons down to many picoNewtons, and can examine a wide range of chemi-cal bonds and processes, such as protein unfolding [7]. This technique uses a precise tip to interact with a sample; however, the presence of the AFM tip and its support-ing cantilever structure within the experiment can introduce unwanted effects on the process under measurement, for example, proximity of a large, charged surface (the cantilever itself).

Magnetic trapping is similar to OT in that an external field is used to control a particle. In the case of MT, this can only be achieved with ferromagnetic and para-magnetic particles, which provides a higher level of selectivity in particle manipula-tion than OT (where unwanted particles are trapped by the laser field) and can be used to provide a constant force on a sample [8]. MT has been successfully applied in the study of DNA uncoiling [9] but is limited to simple spheroidal particles. More complicated structures have been made for use in MT systems but have not been applied for force measurement [10]. A crucial advantage of OT over MT is that the position calibration of an optically trapped object can be made to nanometer accu-racy, which requires the position of the trapping field to be varied in space. This is not the case with MT, where the position of the magnetic field cannot be changed.

FIGURE 12.1 Comparison of Forces in Single Molecule Experiments Measured Using Atomic Force Microscopy (AFM), Magnetic Trapping (MT), and Optical Trapping (OT)

348 CHAPTER 12 Application of optically trapped force probes

The advantages of optical trapping—with flexibility in the spatial distribution of optical traps, optical trap stiffness values, and its minimally invasive nature—make it particularly useful for single molecule force measurement.

1.2 EXAMPLES OF SINGLE MOLECULE OPTICAL TRAPPING EXPERIMENTSExperiments using optical trapping to measure single molecule forces have progres-sively increased in complexity and resolution. The first experiments investigated the extension of DNA tethered to a single trapped particle [11] and the interaction between motor proteins and their filament-like track structures [12]. A classic exam-ple is the interaction of a kinesin protein with a microtubule (Fig. 12.2A), where the protein moves along the filament in a stepping motion [13]. This interaction forms the basis of vesicle transport in cells. Crucial to this measurement is the binding of the protein to a trapped particle and then bringing this to the microtubule where the interaction occurs. Further refinements to this technique involved feedback control of the trapped particle position to provide a constant force against the motion of the protein [14].

To study interactions between proteins and DNA, one end of the filamen-tous DNA strand was tethered to a surface, while the other end was free to move (Fig. 12.2B). One key experiment measured the initial stage of gene expression, known as transcription, where RNA polymerase translocates along a strand of DNA [15]. Importantly, this allowed measurement of the process under study to be made away from the influence of the substrate surface. Further control of DNA was the focus of further optical trapping experiments borrowed from previous expertise using myosin proteins [12,16,17], where the filament was fixed at two points using two optical traps and separate beads (Fig. 12.2C), known as a “dumb bell” [18,19]. An ultrastable setup could be constructed to provide base pair resolution by con-trolling the second trapped particle position using feedback to provide a constant force [20].

One of the most intricate trapping experiments to date has involved the use of DNA as a scanning probe, where it is wrapped around a second piece of DNA that has a protein bound to it (Fig. 12.2D) [21]. This ingenious use of DNA required the use of a complex, automated trapping system and microfluidic technologies to flow in the various components needed to construct the DNA scanning probe and the DNA segment for testing.

All the single molecule experiments shown in Fig. 12.2 rely on the control of a filament structure and the controlled introduction of a single protein. This requires a high level of sample preparation, including specific chemical bonding between the optically trapped particles and the single molecules under study.

In order to confirm that a single molecule is present and interacting as intended, complementary techniques are needed in single molecule force experiments. Advanced forms of fluorescence microscopy can be used here [22], where both the single molecule and the macromolecule that it interacts with (e.g., a DNA strand) are tagged with different fluorescent chemical labels.

3491 Introduction

Typically the optically trapped particles used in single molecule experiments are polystyrene beads with diameters ranging from one to several micrometers. These beads are well suited to single molecule experiments as they are symmetrical and are easily trapped in water due to their size and refractive indices, and they can be supplied with suitable chemical coatings and in high concentrations. This is impor-tant as small differences in experimental conditions can generate variance in results obtained from highly sensitive single molecule experiments.

However, the use of spherical beads as optically trapped force probes does lead to some disadvantages. Exact control over a point on the bead surface is not pos-sible and the bead can spin while trapped. Further, the position of a single molecule attached to a spherical bead cannot be controlled. Also, the surface of the bead where the single molecule is bound will be exposed to the intense trapping field, which will affect the process under study, for example, leading to localized heating [23] or cause reduction in fluorescence activity in experiments that combine force measurement and fluorescence imaging [24]. Chapter 13 discusses the use of laser-heated micro-beads in cellular thermotaxis experiments.

Alternatives to the spherical bead structures have been developed, for example, optically trapped cylinders made from quartz, which allow controlled rotation of the cylinder due to their birefringent optical properties [25]. These cylinders have been successfully applied to twist lengths of DNA [26].

FIGURE 12.2 Examples of Single Molecule Optical Trapping Experiments

(A) An optically trapped bead has a single kinesin protein attached, this walks along a microtubule (shown as a cylinder fixed to the surface). (B) a protein (such as RNA polymerase) fixed to an optically trapped bead which interacts with DNA that has one end fixed to surface. (C) instead of fixing one end of the DNA filament to a surface, each end is attached to a corresponding optically-trapped bead, this is known as a dumb-bell assembly. (D) DNA scanning probe using four traps; one DNA strand is looped around the target strand and scans along in search of bound proteins.

350 CHAPTER 12 Application of optically trapped force probes

To further localize the point of interaction, smaller particles, such as gold nanoparticles, have been optically trapped and the dynamic nature of lipid mem-branes have been measured [27]. Chapter 11 details their use in membrane experi-ments. There are limitations with this approach; first, the trapping efficiency reduces with the particle size due to the overwhelming effects of thermally generated colli-sions in liquid environments. Enhancing the electromagnetic field using apertures or metallic structures [28] offers a static solution to trapping nanoparticles at fixed positions, however, this does not yet offer the flexibility needed for a force measure-ment experiment.

Second, since accurate force measurement relies on position measurement, it becomes increasingly difficult to measure the position of the particle, as size approaches the optical diffraction limit, using the normal approach of bright field microscopy (i.e., observation with using a microscope lamp). Alternative techniques (such as dark field microscopy [29] or total internal reflection fluorescence microscopy [30]) offer the possibility to observe the particles but with much lower signal levels. As we continue in this chapter, we will explore a force probe developed to address the two limitations of reduced trapping efficiency and difficulty in position measurement.

2 FORCE PROBE DESIGNOptically trapped force probes were designed to meet two criteria. First, the probe should have a protruding nanometer scale tip that localizes the force involved in the single molecule interactions. Second, the probe should have a micrometer-sized main structure which serves two purposes: (1) to achieve a high level of trapping efficiency and (2) allow accurate and flexible position measurement when trapped using an optical microscope. An example of a force probe with its protruding tip is shown in Fig. 12.3 and comprises three trapping points, which provide information on the angular position of the probe (i.e., if it is tilted).

FIGURE 12.3 Illustration of a Force Probe Trapped at Three Points

3512 Force probe design

The protruding tip allows the point of force measurement to be placed remotely from the intense optical trapping field, thereby minimizing unwanted effects on the sin-gle molecule under study (such as laser absorption or heating). The intensity of the field is represented by a point spread function (i.e., a Sinc function) that is calculated based on the components used in the optical trapping system (specifically, the microscope’s objective lens). The distance from the maximum to the first minimum in the intensity pattern is represented by an Airy disk [31], with a radius of defined by Eq. (12.2):

λ= ×r

NA

0.61 (12.2)

where λ is the wavelength of the trapping laser and NA is the numerical aperture of the objective lens. For a typical optical trapping system, λ = 1064 nm and NA = 1.2, thereby r = 540 nm. Since r represents the distance to the first minimum in the inten-sity pattern, this value does not represent the entire trapping field [32]. So to take this into account and minimize the intensity at the end of the probe tip, the designed distance of the protruding tip from the nearest trapping field should exceed the value of r by 3 times.

The material from which the force probe is made is an important consideration. The material should have minimal absorption of the trapping laser light and have a refractive index that is not substantially different to that of water (n = 1.33), which is the environment in which it will be used for single molecule experiments. The mate-rial should also be amenable to production processes that produce precise nanometer scale features. These processes are typically found in the microelectronics industry and so the inspiration for the probe material comes from an epoxy-based material called SU8, which is often used in microelectronics production as a photoresistive layer to define microelectronic components [33]. This material has a refractive index of 1.596 measured at a wavelength of 633 nm [34], which meets our criteria. SU8 can be formed into a micrometer-thick polymer layer from which shapes can be etched, and it is a biocompatible polymer, that is, it does not significantly interfere with living biological cells [35] and is fluorescent when excited with blue laser light [36]. These properties make this polymer ideal for single molecule experimentation where the sample is fluorescently labeled, permitting the position of the probe and the fluorescently tagged sample to be observed at the same time.

2.1 PROBE PRODUCTION PROCESSESAmong the range of processes that can be used to make nanometer scale features, electron beam lithography (EBL), and two-photon polymerization (2PP) are the most commonly used [33]. EBL is a standard microelectronics production process, where an electron beam is scanned across a surface to write a pattern that corresponds to the probe shape. This process is repeated many thousands of times to produce an array of 4000 probes (Fig. 12.4A). For our purposes, we produce a layer of SU8 polymer that is several micrometers thick, apply the EBL, and then remove the unexposed parts of the SU8 polymer layer.

r=0.61×λNA

352 CHAPTER 12 Application of optically trapped force probes

A close up of a force probe is shown in Fig. 12.4B, and shows that EBL is a “top-down” production technique that cannot produce rounded structures. In this case, the trapping points for the probe are cylindrical in shape. The optical trapping of cylindrical objects has been demonstrated [13] and it has been shown that they can be trapped in a stable fashion provided that the cylinders have an aspect ratio (i.e., ratio of cylinder height to width) of 1 or greater [37]. The height of the cylinder is determined by the thickness of the SU8 polymer layer that is produced, which in turn puts a limitation on the size of the cylinder width, which must be the same as the height or greater to achieve trapping. For our work, we used a SU8 epoxy layer of 4 µm thickness. This in turn meant that the trapping points were cylinders with a height and diameter of 4 µm. The connecting struts had a width of 200 nm and a depth of 4 µm and the protruding tip of the probe had dimensions of 200 nm wide by 2 µm long by 4 µm deep. The probes shown in Fig. 12.3 are a prototype design, with a protruding tip that ends with an additional gold tip, which was deposited after the initial SU8 deposition step and processed using EBL. Unfortunately, these versions did not survive the release process and so probes with the protruding SU8 tip only were used in single molecule experiments reported later in this chapter.

An alternative production process for the force probe is 2PP, which can make shapes with rounded features. Chapter 2 discusses some basic principles of this tech-nique. This technique uses a focused, pulsed laser beam to selectively polymerize a layer of SU8, which is then solvent exposed to remove the unwanted material and leave the probe shape [38]. This technique is capable of producing many intricate and complex microstructures [39] with feature sizes smaller than 50 nm [40]. An example of a force probe produced using 2PP is shown in Fig. 12.5. This probe consisted of three rounded trapping points with a diameter of 3 µm and a tip that

FIGURE 12.4

(A) Array of force probes produced using electron beam lithography, (B) close-up of a single force probe.

Reproduced with permission from Pollard MR, et al. Optically trapped probes with nanometer-scale tips for

femto-Newton force measurement. New J Phys 2010;12:113056, IOP Publishing & Deutsche Physikalische

Gesellschaft. CC BY-NC-SA.

3532 Force probe design

extended 7 µm from the probe body with a minimum tip width of 200 nm. One draw-back with the technique is that a connecting stub between the probe microstructure and the substrate must be used and then broken to remove the probe. This process can produce variation in the length of the stub at the end of the probe.

2.2 PROBE RELEASE AND LOADING INTO SOLUTIONBoth 2PP and EBL methods require a separation step to remove the force probes from the solid substrate and transfer them into a liquid solution. This operation is fur-ther complicated as the probes need to be collected from an area of several millime-ters square on the substrate. Therefore, to maximize the likelihood that the released probes are collected, the volumes of liquid used in the solution must be kept to a minimum (i.e., less than 1 mL).

The separation step for the EBL method requires application of a droplet of chrome etchant (ceric ammonium nitrate) to chemically remove a layer beneath the force probe. The use of such chemicals, and their acidic nature, can potentially affect later experiments. Therefore, additional steps to neutralize the solution were required. In our case, after 30 min, we remove the chrome etchant and washed the probe area using water several times while checking the pH of the postwash droplets. Finally, the probes could be removed by vigorous pipetting with water. The probes were added to a buffer solution consisting of 50 mM tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 1 mg/mL bovine serum albumin, and 0.01% (v/v) Tween 20. This complete process is reported in Simons and coworkers [41].

The separation step for 2PP involves immersing the force probes in an ultrasonic bath for a short period (less than 1 min). This breaks the stub connecting the force probe to the substrate and releases the probes into solution. The probes are then dried onto the substrate coverslip for transport. Use of the probes in an experiment requires

FIGURE 12.5 Image of Force Probes Created Using Two-Photon Polymerization

Reproduced with permission from Pollard MR, et al. Optically trapped probes with nanometer-scale tips for

femto-Newton force measurement. New J Phys 2010;12:113056, IOP Publishing & Deutsche Physikalische

Gesellschaft. CC BY-NC-SA.

354 CHAPTER 12 Application of optically trapped force probes

the coverslip to be immersed in water and then the probes to be released again using a short exposure in an ultrasonic bath or by applying a pulsed laser beam focused nearby the probe to generate an acoustic shockwave that releases the probe. A typical laser to use in this case would have a pulse duration of <1 ns, wavelength of 1064 nm, and a pulse energy of 10 µJ [42].

The number of force probes produced from one substrate that can be applied in an experiment (example.g., 4000 probes in 0.5 mL solution using EBL) is much smaller than the number of spherical beads that can be delivered in optical trapping experi-ments (as many as 106 beads in 1 mL [43]). This dramatic reduction in numbers means that locating a force probe in an experimental setting (i.e., in a flow cell) can be more difficult than in the case of using spherical beads. One solution to speed up the process of locating a force probe is to create a specialized flow cell that collects the force probes in a predefined area. Our solution involved the development of a flow cell with two channels, one channel that allowed our single molecule experi-ment to be setup and a second channel for the controlled release of the force probes, as shown in Fig. 12.6. The flow cell design can be easily made by cutting double sided tape to define the channels (most double-sided tapes will work, e.g., 3M), with the top and bottom lids of the flow cell made from standard microscope slides and cover slips. To prevent unwanted binding of sample to the microscope slides the surfaces are chemically treated with polyethylene glycol. Ports to allow liquids to be loaded into the flow cell were made by drilling with a diamond tip drill bit (Precision Dental). Force probes were delivered directly to the flow cell via a port that could be resealed using Sellotape after initial testing showed that the probes would be lost by

FIGURE 12.6 Schematic and Photo of Flow Cell Used for Single Molecule Experiments

3553 Experimental setup

binding to the inner walls of the tubing. A low volume gas-tight syringe was used to inject the probes and had a minimal dead volume, thereby limiting the amount of probe solution that was left in the syringe after use, and maximizing the amount of probe solution loaded into the flow cell.

3 EXPERIMENTAL SETUPThe experimental system for the single molecule experiments using the force probe is based around an inverted microscope, as shown in Fig. 12.7. This system com-prises a number of functionalities, flexible optical trapping, and bright-field imaging of the force probe combined with fluorescence imaging of both the force probe and the single molecules under study. This requires a microscope with multiple ports that allow multiple laser beams to enter, for example, in our experimental work we used a Nikon TE2000-S microscope.

3.1 OPTICS AND LASER FOR TRAPPINGA key component of an optical trapping system is the objective lens that provides the strong focusing of the trapping laser light. Chapter 1 discusses basic ideas in

FIGURE 12.7 Schematic of the Experimental System for Single Molecule Experiments

356 CHAPTER 12 Application of optically trapped force probes

optical trapping. When a single laser beam is used, this type of optical trapping is referred to as single beam gradient optical trap, as first discovered by Ashkin and coworkers [44]. Here, a transparent microscopic particle will experience optical forces that push it into the focus of the laser beam, as shown in Fig. 12.8. A balance of two forces hold the particle at the focus; scattering forces which act axially in the direction of laser beam propagation and gradient forces which act toward the focus of the laser beam.

The tight focusing cone of the laser beam, which is described by the numerical aperture of the objective lens, generates gradient forces that balance the scattering forces and act to keep the particle close to the beam focus. The numerical aperture (NA) is defined in Eq. (12.3):

φ=NA nsin (12.3)

Where n is the refractive index of the solution and φ is the half-angle of the arc formed by the focused laser light. Typically, an objective lens with a NA greater than 1.2 and magnification of 60× is used in single beam gradient optical trapping, and requires index-matching oil between the objective lens and the glass coverslip that forms the sample container. At the focal point of the objective lens, the area that can be observed is known as the “field of view,” which is approximately 250 µm in diameter in this case of a 60× objective lens (Olympus UPLSAP060XW). In experi-ments where large sample containers are used, it can be advantageous to use a lower magnification objective lens (10× or 20× magnification) to allow force probes to be located more quickly and then switch to the objective lens used for trapping. For combined fluorescence imaging and optical trapping shown later in the chapter, we used a specialized objective (Nikon 60× APO TIRF, NA 1.49).

NA=nsinφ

FIGURE 12.8 Diagram of Forces Acting on a Particle in a Single Beam Gradient Optical Trap

3573 Experimental setup

The choice of laser beam in optical trapping is dependent on the single molecules under study. If fluorescence labeling of single molecules is used, then it is better to use a laser wavelength greater than the visible wavelengths used for fluorescence imaging, such as a near infrared laser operating around 1 µm. A laser output power of several watts is recommended, as the use of optical components in the system (such as those used to create multiple optical traps) will introduce losses.

3.2 METHODS FOR CREATING MULTIPLE TRAPSThe optical trapping system shown in Fig. 12.7 produces multiple independent opti-cal traps, which allows the force probe to be trapped at the required number of points across its structure. For example, the triangular force probe shown earlier in Fig. 12.4, three optical traps were required. For this system, the component used to create the multiple optical traps is called an acousto-optic deflector or AOD [45]. This com-ponent works on the principle of diffracting an incident beam of laser light using a periodic variation in the refractive index of a block made from tellurium dioxide. The periodic variation is created by sending an acoustic wave across the block and is proportional to the frequency and amplitude of the acoustic signal. In effect, a dynamic diffraction grating is established and the first order of the diffraction pattern is used as the laser beam to produce the optical traps. Using two orthogonally placed blocks allows the AOD to create a two-dimensional diffraction pattern, as shown in Fig. 12.9. Multiple independent optical traps are created by varying the frequency acoustic signals at a rate of kilohertz, which in turn varies the diffraction pattern cre-ated by the AOD. This variation allows a single laser beam to control multiple objects

FIGURE 12.9 Two-Dimensional Beam Steering Using a Dual Crystal Acousto-Optic Deflector, Where a Controllable Variation in the Diffraction Pattern is Indicated by the arrows on the right hand side of the figure

358 CHAPTER 12 Application of optically trapped force probes

by moving between them before the objects move (or diffuse) a significant distance away from the optical trap [46].

Placing the AOD within a conjugate plane of the microscope objective’s rear focal plane [47] allows the angular change in the beam deflection to be accurately relayed to a translation in the image plane of the microscope. AODs have been shown to have very high levels of angular resolution [48] which in turn allows nanometer precision adjustments of the trap position. This level of precision displacement is essential for good control of optically trapped force probes using multiple optical traps.

Alternative optical trapping systems have been developed which use other types of optical devices to create multiple traps (such as holographic trapping [49] and gen-eralized phase contrast [50] which use spatial light modulators [51]). In single beam gradient optical traps, a laser with a Gaussian intensity distribution is used, but laser beams with more complicated spatial modes, such as Bessel beams [52], allow the laser light to be focused over longer lengths extending the range of optical manipula-tion. In addition, higher order Bessel beams have a unique intensity distribution with a null in the center of the laser beam allowing nontransparent particles with a high refractive index to be trapped. A stronger optical trap can be created using more than one laser beam, for example, counter-propagating laser beams can be focused at the same point to create an optical trap [3].

3.3 POSITION MEASUREMENTThe measurement of the force probe position is crucial for accurate forces determina-tions, as defined by Hooke’s law in Eq. (12.1) earlier. The ideal position measurement system should be flexible and allow multiple points on the force probe to be tracked. Imaging cameras consisting of a two-dimensional array of pixels, such as charge-coupled devices (CCDs) [53] or complementary metal-oxide semiconductor (CMOS) cameras [54], have emerged as preferred devices for position measurement of opti-cally trapped objects. These imaging technologies have been compared to existing position sensitive photodiodes used in optical trapping experiments and found to offer flexible imaging and sufficient imaging rates [55]. When these cameras are combined with visible light provided by the microscope (known as bright field imag-ing [56]) and the illumination is set evenly across the field of view (known as “Köhler illumination” [57]), they meet our requirements for position measurement. As cam-era technology has developed, the number of pixels in the two dimensional array has increased to a level of millions of pixels. If we consider the field of view of the micro-scope with a 60× objective lens is 300 µm, a pixel array of 520 × 520 pixels would sample this field of view with 0.57 µm per pixel. The sampling requirement to image micrometer sized particles can be found from the Nyquist criterion [58], which states that the pixel sampling length (0.57 µm) should be at least 2 times smaller than the smallest periodic feature to be imaged. For the case of objects several micrometers in diameter, this requirement is fulfilled.

There are advantages and disadvantages with both CCD and CMOS cameras, and a complete discussion on these technologies can be found elsewhere [59]. Briefly, the

3593 Experimental setup

design of CMOS cameras allow it to operate with lower noise than a CCD camera, however CCD cameras can be augmented to provide higher signal levels (through electron multiplication [60]) which is beneficial for measurements of very low light intensities. The difference between CCD and CMOS cameras is explained by the method of transferring photogenerated electrons from a pixel on the camera. CCD cameras transfer the charge generated by the incident photons on a pixel directly to a special charge-converter unit for conversion to a digital voltage signal. A CMOS camera has a different pixel design which includes a microelectronic circuit which converts the photogenerated charge into a voltage. The pixel voltages can be accessed at a rate of 106 pixels/s using high speed microelectronics [61], where rows of pixels are accessed sequentially (Fig. 12.10). The next step in creating an image is to con-vert the analog pixel voltages into digital values using an analog to digital converter (ADC) [62]. The speed of this process is increased by using multiple ADCs that convert rows of pixel voltages. Finally, these digital pixel values are transferred and stored in computer memory using a combination of software and electronics.

The result of using this technology has greatly increased the number of images that are collected per second, with modern cameras able to capture 10 to 1000’s of frames per second. The higher frame rates are achieved by reducing the number of pixels per images and by defining regions of interest (ROIs), for example, that contain only images of the force probe under measurement. This allows frame rates greater than 104 frames/s to be achieved, with the aim to provide feedback control

FIGURE 12.10 Method for Accessing Rows of Pixels From a Pixel Array in a CMOS Camera

360 CHAPTER 12 Application of optically trapped force probes

of an optically trapped object. Such positional information on the trapped object can be used to reposition the trapping laser beam, thereby minimizing unwanted motion of the object caused by thermally generated collisions in solution [63]. This frame rate is also useful for single molecule experiments permitting time-resolved images of events that occur on millisecond and second timescales. A CMOS camera was chosen for the position measurement of the optically trapped force probe as it could provide faster imaging (>104 frames/s) and provided lower noise levels than a CCD camera. Previous work has demonstrated that the position of multiple optically trapped objects can be measured at a rate of 15 k measurements per second using such a CMOS camera [64].

The position measurement algorithm converts the pixelated image of the opti-cally trapped object into a set of position coordinates. The position measurement can be made by subdividing the image of the force probe into regions of interest (ROIs). In our case, we define three ROIs over the three points of the triangular force probe, as shown in Fig. 12.11.

Each ROI image of the trapping point is essentially a point spread function [65] that is pixelated. Here the number of pixels per ROI should be chosen such that the PSF maps to greater than three pixels which allows the maximum of the PSF to be identified from three points. We chose a ROI size of 6 × 6 pixels, as shown in Fig. 12.11. To optimize the ROI image of the trapping point, it can be necessary to adjust the microscope focus to maximize the height of the PSF. However, when the force probe is optically trapped it is held at the focus of the microscope. Therefore an offset can be added to the position of the CMOS camera that adjusts the focus of the image on the camera.

FIGURE 12.11 Subdivision of Force Probe Image Into Three Regions of Interest for Each Trapping Point of the Probe

3613 Experimental setup

If we use a simplification that the motion of the optically trapped object is within the image plane of the microscope, then orthogonal coordinates for the plane (x and y) for each ROI can be defined. A centroid calculation can then be performed on each ROI to produce a position measurement that is relative to the center of the ROI. This calculation is shown in Eq. (12.4):

∑∑

∑∑= ==

==

C

xI

Ix

ijj

N

i

N

ijj

N

i

N11

11

(12.4)

Where the centroid position in the x direction (Cx) is found from an image of size i by j pixels, by multiplying the pixel values (Iij) per row in the image by the pixel column position in the image (x) and then adding them together, which is then repeated for all the rows in the image (N rows in total). The centroid value is then found by dividing by the sum of pixel values in the image. The centroid position in the y direction (Cy) is then found by replacing the pixel column position (x) by the pixel row position (y).

3.4 ADDITIONAL COMPONENTSAs reported earlier, in single molecule experiments there is a need for complemen-tary measurements that independently identify the presence of a single molecule. The system in Fig. 12.7 uses fluorescence microscopy to identify single molecules. A con-tinuous wave laser with an output of < 5 mW is sufficient for fluorescence imaging, where the laser wavelength is selected according to the fluorescent chemical tags used to label the single molecules under study. A highly sensitive electron multiplier charge coupled device (EMCCD) imaging device is used to detect the low levels of fluores-cence from the labeled single molecules [41]. To reduce the effect of background fluo-rescence, a special variation of fluorescence imaging, called oblique angle fluorescence [66] was used that illuminate the sample at an angle to the microscope image plane instead of directly perpendicular, as is the case in standard epifluorescence imaging.

Dichroic mirrors are optical components that play an important role in this sys-tem, to allow laser light to be sent to the sample but also to prevent any unwanted laser light travelling to the sensitive imaging equipment. The dichroic mirrors are formed from a series of dielectric layers that reflect particular wavelengths and trans-mit the remaining light in other wavelengths [67]. In Fig. 12.7, we use two dichroic mirrors, one to reflect infrared laser light to the sample for trapping and transmit vis-ible light to be sent to the imaging equipment (shown as DM2 in Fig. 12.7). The other dichroic mirror reflects the excitation laser light for fluorescence imaging and trans-mits longer wavelengths (including the emitted fluorescence light). Several blocking filters are used to further attenuate any remaining laser light.

The scanning motion of the optically trapped force probe is achieved by moving the environment around the probe (i.e., moving the flow cell itself). An automated

Cx=∑i=1N∑j=1NxIij∑i=1N∑j=1NIij

362 CHAPTER 12 Application of optically trapped force probes

microscope stage system is used to achieve this effect; the stage can provide move-ment parallel and perpendicular to the optical axis of the microscope. The stage system can be actuated using stepper motors (providing many micrometers of move-ment but with lower repeatability) or piezoelectric actuators (providing lower move-ment region but higher repeatability). Stepper motors were used in the system shown in Fig. 12.7.

4 FORCE PROBE CALIBRATIONThe method for calibrating an optically trapped force probe consists of three stages, initial calibration of laser positioning, calibration of force probe position when trapped, and finally trap stiffness measurements. We describe these three stages and then describe three experiments that measure force with the probe.

4.1 INITIAL CALIBRATION OF LASER POSITIONAn initial calibration of laser position determines the range of laser repositioning in the image plane of the microscope. A calibrated laser position allows nanome-ter precise movement of the optical traps and the force probe. The AOD (shown in Fig. 12.7) continually sweeps the laser along one axis at a rate of 10 kHz. This produces a line when observed with the CMOS sensor that will be used for position measurement, after laser line filters are temporarily removed to allow the laser to be observed. The laser power should be set as low as possible during this work. The line created by scanning the laser position can be measured by the number of sensor pixels that it extends over, then by using the value for the equivalent pixel length in the microscope image (i.e., the pixel length of sensor divided by the magnification of the objective lens), a calibration of the laser position can be made. For example, the AOD used in the work reported here was an Isle Optics TS100-XY AOD with a maximum deflection range of 114 mrad (milliradians), with a bandwidth of 28 MHz and center wavelength of 42 MHz. This produced a line that was 191 pixels long on the CMOS sensor image, corresponding to a maximum deflection of 79.6 µm, based on a pixel sampling of 0.419 µm of the microscope image using a 60× objective lens. The change in AOD frequency for a 1 nm change in laser position can then be found by dividing the bandwidth of the AOD by the maximum deflection length expressed in nanometers:

= = ××

=fBW

L

28 10

79.6 10351Hz / nmstep

6

3defl

(12.5)

This value (351 Hz/nm) is important when calibrating the force probe position. The electronics that produced the AOD drive signal had a frequency resolution of 1 µHz and a frequency stability of 1 ppm (part per million) [68], that is, for an AOD signal with frequency of 42 MHz was stable to 42 Hz, or a positional stability of

fstep=BWLdefl=28×10679.6×103=351 Hz/nm

3634 Force probe calibration

0.12 nm based on Eq. (12.5). This indicated that the signal source was sufficiently accurate to provide the required frequency change.

4.2 CALIBRATION OF FORCE PROBE POSITIONThe position of the optically trapped force probe was calibrated by adjusting the position of the probe relative to a defined region of the sensor image, known as a region of interest (ROI). We used three ROIs, one for each trapping point of the trian-gular probe, where one ROI was 6 pixels long by 6 pixels wide. The ROIs were first aligned over the trapping points of the probe so that the trapping points were centered in the ROI (as shown earlier in Fig. 12.11). At this point, it was important to ensure that the axes of the sensor image were as parallel as possible to the axes of AOD deflection, where the sensor was rotated using a C-mount adaptor on the microscope. This allowed the motion of the probe to be parallel to the sensor images and allow for calibration in two directions across the sensor image.

The force probe was moved in steps of 2 nm in one direction across the ROIs. This movement was achieved using the AOD calibration value calculated in Eq. (12.5) which allowed the corresponding movement of the optical traps. For each step a set of ROI images was taken. This was then repeated for the opposite direction. The ROI images were then analyzed by calculating the centroid values for each trapping point following Eq. (12.4) and the centroid values were plotted as a function of probe position. The centroid values for the three trapping points are shown in Fig. 12.12. A linear region for the centroid measurement was found to be ± 850 nm from the center of the ROIs. All the data for the probe position shown later in this chapter were con-verted to values in nanometers based on the gradient of this linear region.

4.3 TRAP STIFFNESS MEASUREMENTOnce the position of the force probe had been calibrated, the optical stiffness of the probe can be measured. The trap stiffness is represented in the expression for the forces acting on an optically trapped object:

γ+ + =mx t x t kx t F( ) ( ) ( ) rand (12.6)

where x(t) is the position of the trapped object as a function of time. The inertial force is shown as the first term in the equation for the object mass (m), the hydrody-namic force is the second term in the equation involving the viscous drag coefficient (γ), the third term is the optical trapping force with the coefficient “k” being the trap stiffness. The fourth term, Frand, is the force due to random, thermally gener-ated forces on the object when in solution. Inertial forces are much smaller than the hydrodynamic forces as the objects are microscopic and therefore can be neglected. The microscopic trapped object is in a liquid environment and experiences very low turbulence (also described as having a low Reynold’s number [69]) and therefore behaves as an overdamped oscillator, where fast movements of the object are heavily attenuated by the liquid surroundings. By analyzing Frand, then the trap stiffness can

mx(t)+γx˙(t)+kx(t)=Frand

364 CHAPTER 12 Application of optically trapped force probes

be determined. Based on this idea, two methods measure the trapping stiffness of the optically trapped objects based on the variation of its position: power spectrum and equipartition theorem.

The power spectrum method uses Fourier analysis to describe the variation in position of the optically trapped object. This motion is described as an over-damped oscillator, which is represented in the frequency domain by the equation:

π γ=

+S f

k T

f f( )

( )xxB

202 2 (12.7)

where kB is Boltzmann’s constant, T is the temperature in kelvins, γ is the viscous drag coefficient, f0 is the roll-off frequency indicating where motions are damped, and f is the frequency computed from the Fourier transform of the trapped object’s position measurements. This roll-off frequency is visualized in Chapter 1, Section 2. The trap stiffness, k, can be found from values for f0 and γ using the equation:

πγ=k f2 0 (12.8)

The equipartition theorem approach uses the variation of probe position due to ther-mally driven fluctuations created by the collision with water molecules to calculate

Sxx(f)=kBTπ2γ(f02+f2)

k=2πγf0

FIGURE 12.12 Centroid Values for the Three Trapping Points of the Probe, as it is Moved in One Direction (the y Direction)

3654 Force probe calibration

the trapping stiffness of the probe. This involves the same position data as used in the power spectrum analysis. The trap stiffness is calculated from:

=kk T

xB

2 (12.9)

where 〈x2〉 is the statistical variance of the probe position.The viscous drag coefficient, γ, can be affected by the trapped object’s proximity

to a surface (known as Faxen’s law [70]). We avoided this problem by perform-ing the single molecule measurements at a height of 5 µm above the surface of the sample cell; the precise details for this are given in the next section. The viscous drag coefficient is well known for simple objects such as a sphere, but for complicated shapes (such as the force probe) the drag coefficient is more complicated to calculate and requires computer simulation in order to make accurate calculations of the trap stiffness. Chapter 3 discusses these rigorous calculations. Such computer simulation is beyond the scope of this work. However, use of the equipartition theorem did not require calculation of γ and was chosen for its simplicity and its high level of accuracy [71]. We expect that as the force probe has three trapping points that are connected together in a rigid form, then the trap stiffnesses should be similar for the three trapping points on the probe. This was indeed the case and therefore we used the average trap stiffness for the three trapping points for further force measure-ments. In the case of the probe produced using EBL, as described earlier in the chap-ter (to be referred to as the EBL probe), the average trap stiffness was 0.030 pN/nm in the x direction and 0.023 pN/nm in the y direction, for a total laser power of 435 mW measured at the sample.

In addition, we assessed the magnitude of forces that could be sensed with the EBL probe by using an escape force measurement to approximate the magnitude of the force that could be sensed with the probe. The escape force measurement requires a motion to be applied to the surrounding flow cell which induces a hydro-dynamic force on the trapped object. The magnitude of this force (FH) is found using Stokes’ law:

γ=F VH (12.10)

where γ is the viscous drag coefficient and V is the velocity of the motion driving the flow cell. The velocity is increased until the trapped object escapes the trap. Approximating the probe to be a summation of three spheres that were 4 µm in diameter, an approximate value for γ was found to be 1.01 × 10−6 Ns/m. We tested one case where the motion of the flow cell was parallel to the tip and found that a stage velocity of 30 µm/s caused the probe to escape the trap, corresponding to an approximate escape force of 30 pN. This value indicated an upper limit for the opti-cal trapping force that could be applied to the force probe given the maximum laser power applied (435 mW).

The force measurements made with the probe were limited to the image plane of the microscope and no axial force measurements were made. This was due to

k=kBTx2

FH=γV

366 CHAPTER 12 Application of optically trapped force probes

the position measurement technique that did not include measurement in the axial direction. However, an approximation was made by visual observation of the force probe during the experiment to ensure that it did not tilt axially which indicated that the forces under measurement occurred in the image plane.

4.4 EXAMPLES OF TEST FORCES APPLIED TO THE PROBEThe first measurement of forces using the probe was by using another optically trapped object, a microscopic sphere, to push into the tip of the probe. A diagram of the experiment is shown in Fig. 12.13, where a polystyrene sphere with diameter of 5.4 µm (from Interfacial Dynamics Corporation product no. 1-5000) was trapped by creating a fourth optical trap using the acousto-optic deflector as described in Section 4.2. The force probe used in all the experiments reported in this chapter was one created from EBL, as described earlier.

The optically trapped sphere was moved using the AOD in steps of 40 nm in the Y direction until it made contact with the probe tip, as shown in Fig. 12.14, and then continued to displace the probe to maximum of 134 nm. The force exerted on the probe was calculated using a scalar average from the position data for the three trapping points, multiplied by the average trap stiffness for the probe. This scalar average is effectively the centroid of the three trapping points and we used this approach in later analysis of displacement data. The force probe had an average trap stiffness of 0.016 pN/nm in the y direction and this produced a peak displace-ment force equal to 2.13 pN. There was an unwanted displacement in the x-direction of 25 nm, equivalent to a force of 0.4 pN, which indicated that the force from the sphere was not completely perpendicular to the tip of the probe. Moving the optically trapped sphere away from the probe tip permitted the probe to return to its original position suggesting that there were no intermediate trapping points within the range of this measurement.

The minimum force that could be detected by the probe was calculated to be 0.24 pN rms (root mean square value) based on the thermally generated position variation in the probe position (represented by the data between t = 0 s and t = 2 s in

FIGURE 12.13 Experiment to Apply Force From an Optically Trapped Sphere to the Probe Tip

Reproduced with permission from Pollard MR, et al. Optically trapped probes with nanometer-scale tips for

femto-Newton force measurement. New J Phys 2010;12:113056, IOP Publishing & Deutsche Physikalische

Gesellschaft. CC BY-NC-SA.

3674 Force probe calibration

Fig. 12.14). We performed several control tests to examine the effect of the trapping field for the sphere on the probe tip, that is, by moving an empty optical trap in the same motion as performed in the experiment. No significant movement on the probe was observed. In conclusion, this experiment showed that the probe could detect femtoNewton forces applied laterally to its tip.

The second test of the probe for force measurement was the application of force from a fixed object, which was a protrusion that had been created from a layer of plastic tape in the flow cell (which was described earlier in Section 3.3). The force probe was optically trapped and aligned such that only the end of the probe tip would be in contact with the protrusion. Then the flow cell itself was moved in a piecewise uniform, periodic motion over a distance of 17 µm and at a speed of approximately 0.1 µm/s as shown in Fig. 12.15.

As the probe tip made contact with the protrusion, the probe experienced a rota-tional force which caused different levels of displacement for each trapping point. As the three trapping points of the probe were physically linked by the rigid probe

FIGURE 12.14

Probe displacement as the sphere is brought into contact with the probe tip, (A) Displacement in y direction, (B) displacement in x direction. The three trapping points have been offset for clarity.

Reproduced with permission from Pollard MR, et al. Optically trapped probes with nanometer-scale tips for

femto-Newton force measurement. New J Phys 2010;12:113056, IOP Publishing & Deutsche Physikalische

Gesellschaft. CC BY-NC-SA.

368 CHAPTER 12 Application of optically trapped force probes

FIGURE 12.15 Experiment to Apply Force to Probe From a Fixed Object

(A) Image taken of probe and obstruction with direction of probe motion indicated by the arrow, (B) probe displacement in x direction, (C) probe displacement in y direction, (D) resulting force on probe.

Reproduced under Creative Commons license CC-BY from Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

3695 Single molecule force experiment

structure, we assumed that the total change in probe position could be represented as a single spring system that was extended and then returned to its normal posi-tion. As described earlier, the centroid of the displacements for the three trapping points was found and multiplied by the mean of the trap stiffnesses for the trapping points. This result was the magnitude of the force on the probe and is plotted in Fig. 12.15D.

As the probe encountered the obstacle, the force on the probe increased to a maximum until the rotation of the probe allowed sufficient space for the tip to move past the protruding obstacle. The motion was then reversed, with a cor-responding force applied to the probe tip and the resulting force peak shown in Fig. 12.15D. Forward and backward motions produced two different forces on the probe, 6 and 12 pN, respectively. The force peak data suggested that the protru-sion was asymmetrical and deflected the probes by different amounts depending on the scanning direction. The larger force peaks (> 14 pN) produced displace-ments that were at the limits of the linear position measurement range and this indicates the maximum force that can be detected with this particular trap stiff-ness for the probe (an average of 0.03 pN/nm in the x direction and 0.023 pN/nm in the y direction). Interestingly, there were two occasions during a scan where the probe did not return to its original position after moving past the obstacle (see t = 40 s and t = 80 s in Fig. 12.15). On these occasions, a small residual force (< 0.5 pN) was imparted on the probe. This temporary effect may have been caused by a small change to the structure of the obstacle (i.e., deforming in response to contact with the probe) or a temporary chemical interaction between the probe tip and the obstacle. In later experiments we did not observe chemical bonding between the probe tip and the objects it encountered, so this suggests that the forces observed in this case were the result of deformation of the obstacle by the probe tip.

5 SINGLE MOLECULE FORCE EXPERIMENTThe force probe produced using EBL was used in a single molecule experiment, first to measure the tension on a suspended piece of DNA and second to measure the topology of a protein bound to the suspended DNA. In this section, we report the experimental process for suspending the DNA and attaching the protein to the DNA, as well as the experimental force data and discussion of the results.

Bacteriophage lambda DNA (48.5 kbp or 16.5 µm contour length, purchased from New England Biolabs) was used to construct “tightropes.” DNA tightropes were formed inside a flow cell in a series of steps; first, 5 µm diameter silica beads were flowed into the flow cell using a syringe pump (World Precision Instruments). The beads were prior coated with poly-l-lysine (Sigma-Aldrich) which made them attach firmly to the flow cell lower glass coverslip. Subsequently, a solution of DNA was flowed over these coated silica beads. When one end of a DNA molecule con-tacts a silica sphere it attaches, the remainder of the DNA is then extended as a result

370 CHAPTER 12 Application of optically trapped force probes

of solution flow. Where the extended DNA meets a second silica sphere, it will attach and thus form a DNA tightrope. Following the setup of DNA tightropes, flow is only again used to introduce proteins into the flow cell; there is no flow during experimen-tal acquisitions. A full account of the DNA tightrope formation process can be found in the work by Kad and coworkers [22]. The DNA tightropes (several micrometers long and 2 nm in diameter) could be visualized under the microscope using the inter-calating fluorescent dye YOYO-1. This dye was excited using a 473 nm laser (Beck and Hickl) and observed using an electron-multiplying charge coupled device (Andor DV888 EM-CCD) through a long-pass 500 nm dichroic, as shown in Fig. 12.7. As explained earlier in the chapter, the force probe created using EBL was found to autofluorescence at these wavelengths. By checking that the force probe’s image was in the sample plane, we ensured that the trapped probe was positioned orthogonally at the correct height to the DNA tightrope as shown in Fig. 12.16A. The force probe was brought into contact with the DNA tightrope by moving the stage, resulting in the force trace shown in Fig. 12.16D. Force measurements were derived from posi-tional changes of the probe (Fig. 12.16B–C) obtained from brightfield imaging and the CMOS camera, as described earlier.

The flow cell was repeatedly moved backward and forward along the y axis over 11 µm. The tension on the DNA tightrope resisted the probe resulting in displace-ment from the trap and therefore a force. Multiple encounters were used to calculate an average tension on the tightropes of 2.2 pN (Fig. 12.16D). The DNA returned to its original state upon removal of the probe, which indicated that the DNA was unaf-fected by the repeated contact with the probe.

Next, the probe was brought into contact with the DNA tightrope and then moved in a direction parallel to the tightrope over a distance of 6 µm. The scanning motion of the probe along the DNA tightrope produced less than 0.5 pN force on the probe. Repeated scans along the DNA produced similar force profiles which indicated that there was no chemical binding (and resulting resistance) occurring between the probe tip and the DNA tightrope (Fig. 12.17).

In the next experiment, a solution of the DNA repair protein UvrA2 was intro-duced to the flow cell [41]. These proteins were conjugated to quantum dots coated with streptavidin (with diameter ∼ 20 nm and purchased from Invitro-gen, Q10121MP). Upon laser excitation (473 nm wavelength), the quantum dots produced fluorescence which allowed the proteins to be observed using oblique angle fluorescence microscopy. The proteins were diluted to a concentration of 1–3 nM in a solution that consisted of 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl2, 100 mM DTT, and 1 mM ATP. Upon injection into the flow cell, the protein-quantum dot conjugates bound to the DNA tightropes. Where a protein was observed bound to a DNA tightrope, the force probe was brought into contact with the tightrope and then scanned along the tightrope so it came into contact with the bound protein, as shown by the illustration in Fig. 12.18. Initial observation of the protein was made using fluorescence imaging and subsequently force measure-ments were made using brightfield imaging coupled with high-speed and accurate position detection.

3715 Single molecule force experiment

FIGURE 12.16 Detection of Forces From a DNA Tightrope

(A) Fluorescence image of probe being moved into contact with DNA tightrope. (B) Position variation in x direction for the three trapping points, with stage motion shown as dotted line. (C) Probe position variation in y direction, for the three trapping points, with stage motion shown as dotted line. (D) Resulting force on probe.

Reproduced under Creative Commons license CC-BY from Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

372 CHAPTER 12 Application of optically trapped force probes

FIGURE 12.17 Scan of Probe Along DNA Tightrope

(A) Position variation in x direction for the three trapping points, with stage motion shown as dotted line. (B) Probe position variation in y direction, for the three trapping points, with stage motion shown as dotted line. (C) Resulting force on probe.

Reproduced under Creative Commons license CC-BY from Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

3735 Single molecule force experiment

Fig. 12.19 shows that the bound protein acts as an obstacle to the scan of the force probe. As the probe tip encounters a bound protein (indicated by the arrow in Fig. 12.19), it experiences a force that rotates the probe about the tip. After a period of increasing force, the probe tip slips over the opposing protein.

By scanning forward and back over the protein’s location, a series of force peaks was recorded as shown in Fig. 12.20. Specifically in Fig. 12.20, two proteins were encountered during a scan resulting in two peaks during the forward scan, which were repeated as the scan reversed. Partial contact with a third protein was also observed at the end of the scan. The presence of the three proteins was confirmed using fluorescence imaging.

The experiment was repeated for a number of DNA tightropes and the range of peak forces was plotted as a histogram that revealed two force populations

FIGURE 12.18 Illustration of Experiment Using a Single Protein Molecule, Where the Probe is Scanned Along the DNA Tightrope

FIGURE 12.19 The Probe Scans Along a DNA Tightrope and Encounters a Bound Protein, as Viewed Using Fluorescence Imaging

Reproduced under Creative Commons license CC-BY from Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

374 CHAPTER 12 Application of optically trapped force probes

FIGURE 12.20 Example Plot of Forces From Interaction From a Series of Proteins Attached to the DNA Tightrope

(A) Position variation in x direction for the three trapping points, with stage motion shown as dotted line. (B) Probe position variation in y direction, for the three trapping points, with stage motion shown as dotted line. (C) Resulting force on probe.

Reproduced under Creative Commons license CC-BY from Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

3755 Single molecule force experiment

(Fig. 12.21). Despite applying forces up to 13 pN, the proteins under test were not observed to detach from the DNA. One possible cause for the two force populations is the asymmetric distribution of the mass that results from the way that the quantum dot would bind to UvrA2, as indicated by crystallographic structural data [72]. This could produce different obstacles to the motion of the probe depending on which face was in contact with the nanoprobe for a particular sweep. An alternative cause could be that the protein can bind in two structural forms to the DNA, where one form rep-resents specific binding to damaged DNA and the other form represents nonspecific binding to the DNA. However structural studies [73] report that this difference in structure is relatively small and so is unlikely to be resolved. A third possibility is that more than one quantum dot is bound to the protein, resulting in the higher peak force. The quantum dots are considerably larger than the UvrA2 protein and so unwanted accumulations of quantum dots would produce a larger obstacle for the probe to slip past. We tested this possibility by examining the fluorescence images for each force measurement. We attempted to correlate quantum dot fluorescence with peak force. However, the distribution was unchanged, suggesting that the cause of the distribu-tion was not due to the presence of multiple quantum dots. In conclusion, the most likely explanation for the force distribution is the asymmetric distribution of mass for a protein exacerbated by the attachment of quantum dots to one face.

FIGURE 12.21 Histogram of Forces Measured From Protein Interaction With Force Probe

Reproduced Under Creative Commons license CC-BY from Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

376 CHAPTER 12 Application of optically trapped force probes

During the encounter with a protein, the probe rotates (as shown in Fig. 12.22). Since the probe tip extends beyond the triangular body, when it is displaced it will move a distance “d,” but the amount of displacement of the probe trapping point from the trap center is lower (as indicated by “x” in Fig. 12.22). Therefore to calcu-late the difference in force experienced at the end of the tip versus at the vertex (the trapped point) we consider the mechanical work done. Since work is force multiplied by distance the ratio of displacements will provide the ratio of forces thus:

=d

x

F

Ft

(12.11)

Where Ft is the force at the tip and F is the force applied by the traps.Using trigonometric identities for d and x, we can rewrite Eq. (12.11) as:

( )=+

=+

F

F y

y tF

l sin

l sin t

..

. 60

. 60t (12.12)

In the case of this force probe, y = 5.9 µm and t = 2 µm.

= ⋅ =F F F F5.9

7.9therefore 0.75t t

Therefore the force exerted at the tip is reduced by 25% relative to the force as measured using the probe trapping points. The data as shown in Fig. 12.20 were cor-rected by this amount.

dx=FFt

Ft=F.yy+t=F.l.sin60l.sin60+t

Ft=5.97.9⋅F therefore Ft=0.75 F

FIGURE 12.22 Diagram of Force Probe Experiencing Motion at the Tip and on the Probe Body

Reproduced Under Creative Commons license CC-BY from Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

3776 Future directions

There is also the question of how the opposing force of the protein can be expressed as forces parallel and perpendicular to the DNA tightrope. If we con-sider that the probe slips past the protein after rotating maximally ∼15 degrees based on the highest peak force, this leads to the situation in Fig. 12.23. A small force of 3 pN exists perpendicular to the DNA but the majority of the force on the probe remains parallel to the tightrope. The effect of the perpendicular force on the protein is small and not large enough to unfold the protein since protein unfolding events occur in the force regime of several tens to hundreds of pN [74], which is beyond what can be achieved with the force probe. The effect of unwanted forces that were perpendicular to the scan along the DNA was also investigated. To determine if the contribution of the force perpendicular to UvrA2 affected the force distribution shown in Fig. 12.21, we plotted the peak force against the angle of the DNA tightrope relative to scanning direction of the microscope stage (Fig. 12.24). This showed the angle of the DNA tightrope did not affect the force distribution measured with the probe.

6 FUTURE DIRECTIONSThe results in this chapter represent an important contribution toward the appli-cation of optically trapped force probe technology into molecular biology. This achievement required a diverse range of techniques (including optical trapping, biochemistry, and imaging) to be brought together functionally in one experiment. The pioneering steps in this work can be built upon for more sophisticated and fac-ile experiments in the future. For example, one major complication was that after the force probe was introduced into the flow cell it required long distance navigation to a DNA tightrope with a bound protein quantum dot conjugate. This step was often

FIGURE 12.23 Parallel and Perpendicular Forces During Probe Scan

Reproduced Under Creative Commons license CC-BY From Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

378 CHAPTER 12 Application of optically trapped force probes

complicated by occasions where the probe escaped the optical traps while moving between randomly distributed tightropes, which required a new probe to be col-lected if the current probe could not be recovered. This step could be made easier if the force probes could be manufactured directly at a known location in the flow cell, thereby removing the need to manually transfer the force probes to the flow cell which results in a loss of probes through adhesion to interior walls of pipettes and other fluid handling equipment. The use of microfluidic technologies that direct the formation of the DNA tightropes in a predetermined position using active, optically controlled methods [21] could also make the initial setup of the experiment easier to achieve.

The position measurements of the probe were made at high speed (15000 frames/s) and this gives exquisitely detailed information on the interaction with a bound protein at a rate that is approximately 1000 times faster than that possible using fluorescence imaging only to observe the position of the protein (typically 10 frames/s). The gradient of the force peak that is created during the protein interac-tion with the probe tip can be further examined, potentially with Fourier analysis techniques [75] that allow rapid events in the protein interaction to be isolated and potentially separated from the background of thermally generated variations in the probe position. Beyond the probe experiments, high speed imaging offers advantages in measuring the interactions between optically trapped actin filaments and myosin proteins (much like that shown in Fig. 12.2C), where interactions were detected by applying a carrier wave from one bead transmitted through the actin to the other. The amplitude of the carrier wave was reduced when myosin bound to actin, however the detection speed was limited to 10 ms using a 1 kHz sinusoidal wave [76]. Events

FIGURE 12.24 Histogram of Peak Forces Measured Versus Angle of DNA Tightrope

Reproduced Under Creative Commons license CC-BY from Simons M, et al. Directly interrogating single

quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe. Sci Rep

2015;5: 18486 [41].

3796 Future directions

occurring on the submicroseconds timescale are biologically important but difficult to access. Using the present system, a much higher frequency wave could be used to reduce the detection lag time bringing detection closer to the sub-ms timescale. One disadvantage of using the high-speed imaging was that the trapping points of the force probe were imaged as three separate regions and so defining which image region belonged to which trapping point was crucial. An alternative approach could be to image the entire probe structure at a lower frame rate (e.g., 5000 frames/s) and calculate the shape of the probe as a means to determine its motion. This could provide a greater measurement range for the probe displacement and allow more advanced position measurement algorithms to be deployed, for example, tracking the motion of the probe in three dimensions.

The design of the probe with three trapping points was suitable for use in the experiments presented here. The displacement of the probe in the single molecule experiments was primarily a rotation, but some translation of the probe occurred as well (i.e., the motion was not truly rotation around a fixed point). Ideally the probe design could be improved to maximize the rotational movement, for example, using a longer probe tip. A further improvement to the probe would be to chemically bind a protein to the tip of the probe (known as functionalization). This could allow the initial binding process of the protein to the DNA tightrope to be examined using the force probe which would act to control the process.

It was not possible in the single molecule experiment to measure the shape of the quantum dot—protein pair, but future work could investigate producing and applying probes with finer tips. The advantage of the probe technology here is that smaller tip structures (on the order of nanometers) can be produced while retaining the microm-eter scale features that allow optical trapping and position measurement. The effect of scanning the probe tip along a surface could be investigated further to see what effect the tip shape has on the measurement. Tip effects are frequently seen in atomic force microscopy, where the geometry of the tip imparts its own signature on the surface measurement, known as “convolution” [77], and this knowledge could be applied here as well.

The main technique for testing the upper force that can be measured with the probe involves applying a hydrodynamic force, that is, moving the liquid envi-ronment (such as a flow cell) around the probe. In combination with mathemati-cal modeling, this provides a means for determining the extent of the linear force measurement region [78]. Further understanding of this force measurement region is crucial to development of the technique and efforts to improve the analysis of forces on an optically trapped force probe [79,80] can provide a basis for a more detailed analysis of the torque experienced by the probe. This rigorous analysis is described in Chapter 4. As an alternative to applying a hydrodynamic force, DNA could be used to further test the force measurement capabilities of the probe. The physical response of DNA to applied forces under different extensions has been studied extensively [81], and the use of extended lengths of DNA as a metrological standard for length and force measurements has been reported [82]. It would be interesting to investigate what level of femtoNewton scale forces could be detected

380 CHAPTER 12 Application of optically trapped force probes

using the probe. An interesting physics experiment (though not a single molecule experiment) measured femtoNewton forces with optically trapped spheres using a second laser beam to induce a small position change [83]. Our probe technology could be applied here by localizing the small perturbation on the probe tip. The unwanted thermal variation in the probe position acts to limit the minimum force that can be detected, but potentially this can be overcome at certain timescales by using feedback control on the probe position [63]. Finally, the protruding probe tip can be utilized to probe very small forces in between surfaces and objects, such as Casimir forces [84].

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385

CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00013-6Copyright © 2017 Elsevier Ltd. All rights reserved.

Konrad Berghoff, Steve Keller, Wolfgang Gross, Lisa Gebhardt, Holger KressUniversity of Bayreuth, Bayreuth, Germany

CHAPTER OUTLINE

1 Introduction: Cell Migration and Local Stimulation of Single Cells ....................... 3852 Methods ........................................................................................................... 387

2.1 Holographic Optical Trapping ..........................................................3872.2 Fabrication and Characterization of Chemical Microsources ...............3892.3 Generation and Characterization of Thermal Microsources ..................3912.4 Automated Cell Shape Detection .....................................................394

3 Results ............................................................................................................. 3973.1 Chemotactic Response to Local Chemical Gradients ..........................3973.2 Response to Local Chemical Perturbations .......................................3983.3 Thermotactic Response to Local Thermal Gradients ...........................400

4 Summary, Conclusions, and Outlook ................................................................... 403Acknowledgments .................................................................................................... 406References .............................................................................................................. 406

1 INTRODUCTION: CELL MIGRATION AND LOCAL STIMULATION OF SINGLE CELLSDirected motion plays a significant role for a large number of cell types. Muscle contraction and relaxation regulated by the mammalian musculoskeletal system [1], wound healing involving migration of tissue cells toward the site of injury [2], synapse formation in the central nervous system [3], immune cell recruitment as part of the immune response [4] always involves motion of cells. Multicellular organisms start their life cycle in an embryonic few cells state, where directional cell migration plays an important role for the subsequent development [5]. Certainly, cellular motion is essential for various facets of life as we know it.

How do cells know where to go? Cells are able to sense their environment physi-cally and chemically and can therefore adapt to local changes of their surrounding by

Application of optical tweezers for biochemical and thermal cell stimulation

13

386 CHAPTER 13 Application of optical tweezers

altering their migration. This way of cellular motion is called taxis, which can have many different characteristics, such as sensing of temperature (thermotaxis) [6,7] or concentration (chemotaxis) changes [8,9] or as reaction to varying light (phototaxis) [10] or stiffness conditions (durotaxis) [11] of their environment. The possibility to apply well-controlled local thermal, chemical, optical, and mechanical stimuli to single cells allows investigating these taxes by monitoring the subsequent cellular responses. Methods of spatiotemporal optical pattern generation and micromanipu-lation are useful tools for applying such local stimuli to cells in a well-controlled manner [12–19]. Optical traps, also named optical tweezers, in particular allow non-invasive stimulation of targeted single cells, thereby offering precise control not only over the nature of the stimulus, but also over its location, duration, and strength. Such applications of optical trapping will be discussed in this chapter with particular focus on thermal and biochemical cell stimulation.

Thermotaxis describes the ability of cells or organisms to sense and respond to thermal gradients in their environment, whereas, chemotaxis denotes the ability of cells to sense and respond to chemical gradients in the environment [6–9]. Thermo-taxis and chemotaxis can be discriminated into three different categories: (1) positive taxis (the cell or organism is moving in the gradient direction); (2) negative taxis (the cell or organism is moving against the gradient direction); or (3) moving along respective isotherms or isochors. Thermotactic motion along isotherms can be ob-served, for example, in the nematode Caenorhabditis elegans [20] or the amoebae Dictyostelium discoideum [21]. Obviously, for organisms which lack active thermo-regulation, thermotaxis is essential to cope with changing temperature conditions in their environment. Thermotaxis is also highly relevant at the single cell level, which can be seen, for example, in the case of inflammation, the body’s tissue response to pathogens like viruses, bacteria, or parasites [22–24]. A characteristic symptom of an inflammation is an increased temperature transforming the site of inflammation into a local heat source for immune cells thermally sensing their environment [23].

Chemotaxis also plays a pivotal role in the case of inflammation [8,25,26]. In-flammation typically is accompanied by the release of chemoattractants, which stim-ulate the innate immune response [22]. This makes the study of local stimuli to cells highly relevant because, depending on the nature of the biochemical stimulus, cellu-lar reactions can be completely different. T-cell expansion for instance is significant-ly increased if cytokines for antigen-specific T-cell stimulation are released directly at the site of interaction with T lymphocytes [27]. In contrast, exogenous cytokines addition to the complete T-cell suspension results in a less effective stimulation [27].

Cellular sensing of molecular gradients is also involved in cell differentiation during embryogenesis [28], wound healing by fibroblast cells [29], or cellular food retrieval [9]. A fascinating example highlighting the significance of chemotaxis can be observed when putting Dictyostelium amoebae under starvation conditions. At nutrient depletion, each single Dictyostelium cell releases chemical signals that can be sensed by proximate Dictyostelium cells resulting in the formation of a multicellu-lar “fruiting body,” which then slings away the top-most cells like spores to possibly reach regions which are richer in food [30]. As a result of its role in a large number of

3872 Methods

eukaryotic processes, chemotactic cell motion has been subject of different models [31,32]. The application of precise local stimulations and the quantification of the subsequent cellular responses are useful approaches to test such models. This chapter highlights some recent advances and experimental approaches for biochemical and thermal cell stimulation with the help of optical tweezers. Interested readers may review the fundamentals of optical tweezers in Chapter 1.

2 METHODS2.1 HOLOGRAPHIC OPTICAL TRAPPINGAll experiments and measurements concerning biochemical and thermal cell stimu-lation presented here were performed using holographic optical traps (HOT) [33–36] because HOT provide precise real-time control over multiple microparticles in live cell experiments [37] and, consequently, similar control over the number, location, and duration of targeted cell stimuli, first published by Kress and coworkers [12]. This enables the investigation of single cellular responses to spatially and temporally flexible and complex microsource geometries. One experimental application of HOT for live cell measurements is illustrated in Fig. 13.1.

Here, the successive phagocytic uptake of several target particles by mouse mac-rophage cells is induced and investigated. The basic HOT principle is depicted in Fig. 13.1A. The trapping laser of the optical tweezers setup is phase-modulated by a spatial light modulator (SLM) located at the Fourier plane of the microscope objec-tive and displaying a computer-generated phase-pattern on its nematic liquid crystal display. In this case, the SLM phase-mask, which transforms the incoming collimated Gaussian laser into a multiple spot geometry, is calculated and displayed as an 8-bit gray value image, where different gray values denote the degree of induced phase shift between 0 (0) and 2π (255). Phase-mask calculations can be done in real-time via a custom-made program code based on a “lenses and gratings” algorithm [34,38], enabling independent three-dimensional steering of each trap and activation of single traps during a measurement. This is pivotal for long-term live cell experiments be-cause it allows reactions to cellular changes without the need to change the field of view during a measurement simply by adjusting the trapping pattern. A typical phase pattern (with a resolution of 512 × 512 pixels), which generates five spots of the trapping laser in the trapping plane inside the microscope (Fig. 13.1C), is shown in Fig. 13.1B. Using this trapping geometry enables simultaneous optical trapping of five polystyrene microparticles (Fig. 13.1D), which are used for a series of phagocy-tosis experiments, sketched in Fig. 13.1E–H. Due to prior surface functionalization of the polystyrene beads with Immunoglobulin-G antibodies, which specifically bind to the Fc-γ membrane receptors of macrophage cells [39], these target beads can be attached to the cell membrane. After establishing cell-bead contact, a series of intra-cellular signaling events, which are triggered by the membrane receptors, allows the cell to engulf its target [39,40]. This process of target recognition and subsequent internalization followed by cellular degradation of the target is called phagocytosis

388 CHAPTER 13 Application of optical tweezers

3892 Methods

[41]. Phagocytosis is a key process of the mammalian immune response in the de-fense of the organism against invaders. Phagocytosis can be found in many different cell types, most prominent are the so-called professional phagocytes like neutrophils, dendritic cells and macrophages [42]. In the example presented here, mouse macro-phage cells of the J774A.1 cell line were used for the determination of the timescales of phagocytic uptake. Fluctuation measurements enable the determination of the time point of binding between a particle and a cell with a precision of about 100 ms [43] allowing precise measurements of the local cellular kinetics following the binding event. The possibility of realizing multiple optical traps with HOT provides a large degree of flexibility in the experiments allowing, for example, the investigation of a sequence of uptake events by offering consecutive target beads to the cell in a row. Furthermore, sufficiently strong HOT enable the measurement of cellular forces and stiffnesses [37], which makes HOT a valuable and versatile tool for live cell studies. Force calibration of HOT can be done by using standard force calibration techniques [44,45], as discussed in Chapter 1.

2.2 FABRICATION AND CHARACTERIZATION OF CHEMICAL MICROSOURCESChemotaxis experiments require control over chemical gradients. Among others, this can be realized by using micropipettes [46], microfluidic devices [47], or by using optically manipulated microsources releasing chemical drugs [12]. The lat-ter approach enables the generation of temporally, spatially, and chemically highly flexible molecular concentration patterns. Depending on the nature of the released chemical, the microsources can act, for example, as chemoattractants or they can cause cell perturbations. Both applications are highlighted in this chapter. Drug

FIGURE 13.1 Holographic Optical Trapping (HOT) Application for Live Cell Experiments

(A) Sketch of the main parts of a HOT system: the trapping laser is phase modulated by a spatial light modulator (SLM) located in the Fourier plane (SLM plane) of the microscope objective (MO). The MO transforms the phase-modulated light from the SLM plane into an intensity pattern on the trapping plane via an optical Fourier transformation, (B) Picture of a typical phase pattern with a resolution of 512 × 512 pixels corresponding to the SLM dimensions. The different gray values visualize the degree of induced phase shift between 0 (black) and 2π (white), (C) Resulting laser intensity pattern on the trapping plane for the phase pattern shown in panel (B) (scale bar 10 mµ ), (D) Image of five simultaneously trapped of target beads (polystyrene, diameter d 2PS = µm) using the trapping pattern shown in panel c) (scale bar 10 mµ ), (E–H) Differential interference contrast (DIC) microscopy image series of a typical HOT application investigating phagocytosis. By using HOT, optically trapped antibody-coated microspheres can be successively attached to a macrophage cell which, in turn, starts to internalize these target objects (scale bar 10 mµ ). Optical traps additionally enable force and stiffness measurements during the phagocytic uptake.

10 µmdPS=210 µm

10 µm

390 CHAPTER 13 Application of optical tweezers

release by the microbeads can be achieved by using various types of materials [48], including biodegradable polyester particles [49] or recombinant spider silk particles [50], which find widespread applications for medical and therapeutic drug deliv-ery [51]. For the experiments discussed here the biodegradable nontoxic copolymer poly(lactic-co-glycolic acid) (PLGA) was used. PLGA degrades by bulk erosion and hydrolysis [52], thereby releasing previously incorporated materials of choice, such as small molecule drugs, proteins, peptides, fluorescent dyes, etc., into the suspen-sion [49]. PGLA microspheres are still optically trappable due to their refractive index mismatch from water [53].

The fabrication of PLGA microsources can be accomplished by using a solvent evaporation–spontaneous emulsion technique [49] as described previously [12]. Briefly, the drug of interest, which was formyl-methionine-leucine-phenylalanine (fMLP) for the chemotaxis experiments discussed here and cytochalasin D (CytoD) for the cytoskeletal perturbation assays discussed here, was added to a solution of the PLGA in dichloromethane (DCM). Then the PLGA solution was added drop-wise under vortexing to an aqueous solution of polyvinylalcohol (PVA). The mixture was sonicated and the resulting oil-in-water droplets were hardened by evaporating the DCM. Resulting microparticles typically show bead diameters between 0.2 and 2 µm and an average polydispersity (standard deviation of diameter divided by mean diameter) of about 40%. The size distribution of the microbeads was determined with scanning electron microscopy (SEM) and with dynamic light scattering [12].

The concentration profile around a microsphere of radius a releasing a drug is controlled by diffusion and can therefore be calculated by using the diffusion equa-tion. As described in [12] in the easiest case, assuming spherical symmetry and steady state, the diffusion equation reduces to ρ( )∇ =c 02 . The solution of this equa-tion yields a concentration c(ρ) with

ρρ( ) =⋅

cc a

,0 (13.1)

where c0 denotes the concentration at the surface of the microsource and ρ the radial distance to the bead center. The situation is more complicated close to a coverslip, where experiments on adherent cells are typically performed. In that case, an ap-proximation of the concentration profile for the microsource, located at a height h above the coverslip, can be done by adding an imaginary microsource located at the negative height −h to the calculation, as described in [12]. The concentration c0 on the surface of the microsource

= ⋅c j aD0 0 (13.2)

can be obtained via the flux j0 from the bead and the diffusion coefficient D of the molecules. The flux j0 can be quantified experimentally by measuring the release of the molecules from the microbeads, for example, with ultraviolet (UV) fluores-cence spectroscopy [54] or high-performance liquid chromatography techniques (HPLC) [55]. The microsources which were used for the chemotaxis experiments

∇c2ρ=0

cρ=c0⋅aρ,

c0=j0⋅aD

3912 Methods

presented here were shown [12] to have a surface concentration c0 in the order of 1–10 nM above the background concentration of the solution, which is sufficient to induce chemotaxis in neutrophils. Generation of larger sources resulting in even higher gradients can be done optionally by optical trapping larger or multiple mi-crobeads inside a single optical trap. The flux and therefore also the concentrations were shown to scale with the volume of the particles [12]. Chemically loaded PLGA microsources show rapid release characteristics during the first 24 h after loading, followed by a slow release period after this initial burst [56]. Therefore, the experi-mental time for the controlled release experiments presented here was limited to last up to about 1 h [12].

2.3 GENERATION AND CHARACTERIZATION OF THERMAL MICROSOURCESThe heating of an optically trapped microparticle and its surrounding medium is in general an unwanted side effect of optical tweezers [57,58]. However, this heating can also be exploited in certain applications, for example, as discussed in Chapter 11. Here, we show how to exploit this effect to generate strongly localized heat sources to create thermal gradients on small length scales. Strongly absorbing materials, like gold nanoparticles [58,59], can, for example, be used for targeted heating in photo-thermal therapies [60], for tuning DNA binding kinetics [61] or for enhancing sur-face plasmon resonance sensing [62]. If laser light is tightly focused, which is crucial in optical trapping applications, the absorbed energies can become very high due to large power densities in the focal plane [57]. For instance, a laser beam with a power of 100 mW, when focused through a high resolution water immersion objective with a high numerical aperture (N.A.) of N.A. = 1.27, already generates a power density of ≈λD 10MW/cm2, which is 8 orders of magnitude higher than the power density of sunlight hitting the earth’s surface ( ≈D 100mW/cm2) [57,63]. This is why, whenever it comes to optical trapping applications with living cells, wavelengths in the near infrared regime are typically chosen, since cells absorb in general less energy in this spectral range than in the visible range and are hence suffering less optical dam-age [64,65]. However even when using near infrared trapping lasers and dielectric particles like polystyrene beads, laser-induced heating of the trapped particles still occurs [57]. As a result, cells in the vicinity of the heat source can react to the thermal stimulus by undergoing thermotaxis. Therefore, optically trapped microspheres can be used for controlled activation of thermotactic cell motion, as will be shown in the following section.

For the thermotaxis experiments described here, plain polystyrene microspheres with diameter of = µd 3 mPS were chosen as thermal microsources. These beads can easily be trapped with optical tweezers because of their large refractive index ( ≈n 1.6PS ). For a quantification of the heating of the beads and the calibration of the thermal gradients around the microsources inside the sample chamber, temperature-dependent fluorescent dyes are useful tools [66]. Such dyes have advantages over bulky thermosensors, like thermocouples or semiconductor thermistors, because they

Dλ≈10 MW/cm2

D∇≈100 mW/cm2

dPS=3 µm

nPS≈1.6

392 CHAPTER 13 Application of optical tweezers

can be used in closed sample chambers, which are necessary to avoid evaporation during long-time cell measurements. The principles of the thermotaxis experiments (Fig. 13.2A) and the temperature calibration (Fig. 13.2B–E) are shown in Fig. 13.2.

The temperature calibration depends on the use of two fluorescent dyes, from which at least one needs to have a fluorescence intensity which is temperature-dependent [66]. Rhodamin B (RhB) and Rhodamine 110 (Rh110) are a suitable pair of dyes. Both dyes have similar molecular structures but show emission spectra max-ima which are sufficiently separated from each other, enabling separate detection of both emission wavelengths within different fluorescence channels. RhB shows a sig-nificantly stronger temperature-dependence of its fluorescence (2.3%/ K) compared to Rho 110 (0.13 %/K) [66] and therefore acts as the indicator for the temperature, whereas Rh110 acts as the reference dye. Fig. 13.2B shows the ratio of the fluores-cence intensity of RhB divided by the fluorescence intensity of Rh110 and normal-ized to the ratio at a reference temperature of =T 310 Kref . The relation between the temperature T and the normalized fluorescence ratio serves as temperature calibra-tion and can, in first approximation, assumed to be linear.

The temperature was controlled by heating an incubation chamber that encloses the whole microscope including the fluorescent sample. From Fig. 13.2B, it can be seen that the fluorescence intensity ratio decreases, in good approximation, linearly with an increasing temperature of the sample. Using this calibration, the temperature gradient around optically trapped polystyrene beads was determined as shown in Fig. 13.2C–E. Polystyrene beads (diameter, = µd 3 mPS ) were dispersed in a mixture of the two rhodamine dyes and optically trapped with a 40× water immersion ob-jective ( =N.A. 1.15) by a laser (λ = 1064 nm ) with a power of ≈P 900 mW in the trapping plane. Fig. 13.2C–D show the resulting fluorescence images of a trapped bead for Rh110 (Fig. 13.2C) and RhB (Fig. 13.2D). One can clearly see the de-creased intensity distribution close to the microbead in the RhB-channel compared to the more uniform intensity distribution depicted in the Rh110 reference channel. Fig. 13.2E shows the resulting temperature relation T(ρ) as function of the radial distance ρ from the bead center. If the bead is trapped sufficiently far away from the bottom surface of the sample chamber (i.e., the coverslip), boundary effects can be neglected and the fluorescence intensity distribution can be assumed to be rotation-ally symmetric and to follow the relation

ρρ( ) = − +I c I ,0 (13.3)

where I0 denotes the background intensity far away from the bead center and c is a scaling factor [12]. If the bead is trapped close to the coverslip, which is in general the case in experiments with adherent cells, boundary effects might not be negligible and the temperature distribution is no longer rotationally symmetric. The closer the bead is to the coverslip, which acts like a heat sink, the smaller the bead heating will be [57]. Shear flow influenced by convection around the heat source was quanti-fied by tracking tracer particles inside the sample. Such particles reached average velocities in the order of 1µm/s , yielding shear stresses which lie about 4 orders of

2.3 % /K0.13 %/K

Tref=310 K

dPS=3 µm

N.A.=1.15λ=1064 nm

P≈900 mW

Iρ=−cρ+I0,

1 µm/s

39

32

Methods

FIGURE 13.2 Generation and Characterization of Localized Heat Sources for Thermotaxis Studies

(A) Sketch of a typical thermotaxis experiment. A cell in the vicinity of the optically heated microsource experiences a thermal gradient. The color scale from red to yellow depicts qualitatively the temperature distribution around the microsource, (B) Quantification of the temperature-dependent fluorescence of selected fluorescent dyes. Shown is the normalized ratio of the fluorescence intensities of the dyes Rhodamine B (RhB) and Rhodamine 110 (Rh110) as a function of the temperature. In a first approximation, the temperature dependence can be considered to be linear (red fit line), (C–D) Microscopy images of the fluorescence intensity distribution around a trapped microparticle with a diameter of d 3PS = µm. It can be seen that the reference dye Rh110 shows a homogeneous fluorescence distribution and can be considered, in a good approximation, as temperature-independent, whereas the dye RhB shows a strong temperature-dependence with suppressed fluorescence in close vicinity to the particle (scale bar 10 mµ ), (E) Application of the temperature calibration data shown in (B) to the ratio of the images shown in (C) and (D) yields the temperature around the microparticle as a function of the distance to the particle. The laser used to trap the particle (power, P 900≈ mW in the trapping plane) also induces heating of the bead of about 14 K relative to the background temperature which yields a temperature increase of about 16 K/W.

dPS=3

10 µm

P≈90016 K/W

394 CHAPTER 13 Application of optical tweezers

magnitude below typical stress values in blood vessels [67]. Cell response to local flow was therefore regarded as unlikely.

2.4 AUTOMATED CELL SHAPE DETECTIONTo quantify the behavior of cells and to obtain parameters describing individual cells, the cells can be tracked individually either in real time during the experiment or during post processing. While manual tracking of the cell position is possible for small datasets it becomes more and more tedious as the number of images and the number of cells per image grow. In particular, manual determination of the cell outline, which is necessary for area and shape analysis, is particularly time consuming.

Automated detection of the cells’ outlines requires input images with good con-trast between the cells and the background. Differential interference contrast (DIC) microscopy is a technique that transforms differences in optical path length through adjacent regions in the sample into differences in intensity [68]. An overview of this technique has been published by Murphy and Davidson [69]. As shown in Fig. 13.3A, DIC provides superior contrast over bright field transmission micros-copy (Fig. 13.3B) where differences in intensity arise mainly due to absorption in the sample.

The application of DIC microscopy in combination with an objective with a high magnification and a high N.A. gives the possibility to visualize individual membrane

FIGURE 13.3 Comparison of DIC Imaging and Bright Field Imaging

(A) DIC image of J774A.1 mouse macrophages. Due to the nature of DIC microscopy the cells are imaged with better contrast, and in more detail, compared to the bright field image of the same cells (B) enabling for instance the imaging of the cell filopodia. The characteristic brightness differences of the DIC image result from optical path length differences due to refractive index or sample thickness changes between adjacent regions of the sample (scale bar 10 mµ ), (B) Bright field image of the same cells.10 µm

3952 Methods

extensions such as lamellipodia and filopodia. Therefore, DIC microscopy was used for all cell experiments described in this chapter.

Due to the nature of DIC microscopy, the edges of the cells appear darker and brighter than the background on opposing sides [69] (Fig. 13.3A). Therefore, simple image thresholding is not a suitable technique to detect the cells. Different tech-niques, such as line integration [70], local entropy detection [71], and variance fil-tering in combination with directional integration [72] have been proposed for au-tomated analysis of DIC images in cell assays. The algorithm used here is based on the detection of areas of high local contrast, which is similar to an approach that has been described for phase contrast microscopy [73]. To quickly adopt the algorithm, extending an existing image processing library is recommended. Here, the code is implemented in MATLAB (The MathWorks, Inc.), making use of built-in-functions, such as standard mask operations and object detection.

The algorithm is summarized in Fig. 13.4. Fig. 13.4A shows a DIC image of an HL-60 cell adherent to a coverslip. While the background is an area of homogeneous in-tensity, high local contrasts inside the cell and at the cell boundary are very prominent. To segment the cell from the background, a small circular area around every pixel in the image (Fig. 13.4B) is scanned. If the difference ( ) ( )∆ = −I I Imax mincircle circle circle

of the maximum ( )Imax

circle and the minimum gray value ( )Imin

circle in the area is

larger than a user-defined threshold, the center pixel of the binary cell mask is set to 1, otherwise to 0:

=∆ >∆ ≤

cellMask

I tresholdI treshold

1,if0,ifij

circle

circle (13.4)

The calculation of the individual cell mask pixels can be parallelized for efficient usage of modern multicore machines. To make the detection less prone to camera noise, a small Gaussian filter with a width (standard deviation) of 1 pixel can be ap-plied before the filtering or the size of the scanned circle can be increased to increase pixel statistics. The result of the contrast detection with a circle radius of 3 pixels is shown in Fig. 13.4C. In this example, most of the cell has been detected by local contrast filtering. However, it turns out that the complete detection of the lamellipo-dia, which are very thin and therefore sometimes barely visible in the DIC images is challenging with local contrast detection alone. Only the outline of the lamellipodia gets recognized reliably. Therefore, the remaining holes (black arrows in Fig. 13.4C) are filled to remove those defects in the mask (Fig. 13.4D). In MATLAB, this can be done with the imfill function.

The outline of the mask is quite rough and contains small areas of high local contrast in the background. To smooth the mask, a Gaussian blur with a standard deviation of a few pixels is applied (Fig. 13.4E). To improve the runtime perfor-mance, the Gaussian filter can be approximated with sufficient quality by a series of box blurs [74]. The filtered mask is then thresholded (Fig. 13.4F). The larger the threshold, the tighter the cell mask gets. Proper threshold values depend on the

∆Icircle=maxIcircle−minIcircle

maxIcircle

minIcircle

cellMaskij=1, if ∆Icircle>treshold0, if ∆-Icircle≤treshold

396 CHAPTER 13 Application of optical tweezers

FIGURE 13.4 Segmentation of a Single Cell in a DIC Image

(A) DIC image of an HL-60 cell (scale bar 10 mµ ), (B) Detection of local contrast is done by scanning a circular region around every pixel for local maximum and minimum gray values (see main text), (C) Result of local contrast detection, (D) Small holes [i.e., black regions that are not connected to the border of the image, see arrows in (C)] are filled, (E) A smoothing filter (e.g., Gaussian blur) is applied to smooth the mask, (F) The filtered mask is thresholded to get a binary image. The threshold can be used to tighten or widen the selection and to remove regions of high background noise, (G) Small objects, such as dust or particles are removed from the cell mask, (H) final detected outline of the cell. The orientation of the cell is defined as the angle between the major axis of the cell and the positive x-axis (blue lines), (I) Result of the local contrast detection algorithm for an image containing multiple J774A.1 macrophages. Distinguishing multiple cells in direct proximity (purple outline) requires the use of more complex algorithms [75] or additional information, for example a stained nucleus (scale bar 20 mµ ).

10 µm

20 µm

3973 Results

radius of the scanned circle and the size of the Gaussian filter. The Gaussian filter also acts as a long pass filter removing small areas of high noise in the background detected by the local threshold filter. This emphasizes the need to fill the mask. When a Gaussian filter is applied to the thin boundary of the lamellipodia, the boundary would get filtered away and the lamellipodia would be left undetected. Any remaining small objects, such as dust or polystyrene beads can finally be removed from the mask (Fig. 13.4G). In MATLAB, this can be done using the built-in function bwareaopen.

Problems arise when polystyrene particles are close to the cell outline. Polysty-rene particles are areas of high local contrast and will also be detected by the routine. We therefore replace particles in the input image with circular discs having the same gray value as the background before the detection.

Fig. 13.4H shows the result of the detection algorithm which is in excellent agreement with the original input data. The center of mass (red cross) and other parameters such as the surface area or the orientation of the cell can then be ex-tracted from the mask. We define the cell orientation ( )P tcell as the angle between the x-axis and the major axis of an ellipse which has the same second moments as the cell. The orientation can take values between 0 and 360 degrees, with respect to the positive x-axis. The MATLAB-function regionprops is suitable for that purpose.

Fig. 13.4I shows the results of the algorithm performed on a DIC image con-taining many J774A.1 macrophages. Individual cells can be separated as long as they are further apart than the size of the scanned circle. Distinguishing multiple cells touching each other is difficult using this algorithm alone but can be real-ized with cell feature detection [75]. Furthermore, the use of additional informa-tion, such as a stained nucleus can be helpful. For the experiments described here, seeding the cells sparsely so that they do not touch and influence each other is preferable.

3 RESULTS3.1 CHEMOTACTIC RESPONSE TO LOCAL CHEMICAL GRADIENTSChemotactic cell response to local chemical gradients can be induced by optically trapped microsources loaded with a suitable drug. In the experiments shown here, PGLA microbeads were loaded with either formyl-methionine-leucine-phenylalanine (fMLP) or cytochalasin D (CytoD) to stimulate cells chemotactically or to induce local cytoskeletal perturbations, respectively. fMLP is a peptide which binds to G-protein receptors which induce chemotaxis as part of the innate immune response [76]. CytoD is a fungal toxin that inhibits actin polymerization by binding to the barbed ends of filamentous actin which, thereby, influences the mechanical properties and the morphology of cells [77]. Visible cellular changes upon CytoD application depend on the drug concentration and can range from cell ruffling and inhibition of cell motility up to the loss of distinct cell shapes due to complete actin disruption [78].

Pcellt

398 CHAPTER 13 Application of optical tweezers

The cells used in the experiments discussed here were from the HL-60 human promyelocytic leukemia cell line which can differentiate into neutrophils [12]. HL-60 neutrophils are well established as a chemotactic model system [46]. The samples for the experiments shown here were prepared by plating the HL-60 cells on fibronectin-coated coverslips and by incubating them at 37°C prior to the microscopy experi-ments. A typical experimental realization of biochemically induced cell migration is shown in Fig. 13.5 [12].

The optically trapped microsource was loaded with fMLP and generated a spa-tially and temporally flexible concentration gradient inside the sample [12]. The fMLP gradient induced direct polarization and migration of the HL-60 neutrophil, which is shown in Fig. 13.5A–F. The trapping powers used in the chemical stimula-tion experiments were about 5 mW, resulting in a negligible temperature increase of the trapped source and its environment, to avoid crosstalk from thermotaxis [12]. The microsource was moved to the cell membrane up to a distance of about one bead diameter (Fig. 13.5A) causing the cell to polarize and migrate toward the bead (Fig. 13.5B). Subsequently moving the particle with a sufficiently low speed leads to cell migration in the direction of the chemical gradient, that is, toward the bead (Fig. 13.5C–F). Quantitative data analysis was performed by tracking the cell with the custom-made automatic cell shape detection algorithm, as depicted in Fig. 13.5A–G. Trajectories of the microsources were determined using a custom-made bead track-ing routine providing subpixel precision of the bead position. Cellular properties like the center of mass,

ρ ( )tcell , the surface area, A(t), and the cell orientation, ( )P tcell , were calculated, as described in the section on automated cell shape detection. The

cell elongation, ( ) ( )( )=E t

C t

A t, is defined as the ratio of the cell perimeter, C(t), and the

area, A(t), while the instantaneous cell velocity is defined as

ρ( ) =v td

dtinstcell . The gra-

dient orientation, ( )P tgrad , denotes here the angle of the vector ρ ρ ρ( )( ) ( )∆ = −t tbead cell

between cellular center of mass and bead center with respect to the positive x-axis and is defined in the interval of ( )< <P t0 degree 360 degreegrad . The plots of

( ) ( )P t P tandgrad cell as functions of time, which are depicted in Fig. 13.5H, quantify the relation of the gradient direction and the cell migration direction. It can be seen that the cell orientation follows the gradient direction. Additional movies of chemotactic cell response, which elucidate the high degree of freedom provided by HOT medi-ated cell stimuli, can be found online [12].

3.2 RESPONSE TO LOCAL CHEMICAL PERTURBATIONSBesides chemotactic cell migration, optically trapped microsources can also be used to investigate other types of highly localized perturbations to single cells. Expos-ing selected cellular regions to well-controlled biochemical stimuli can be used, for instance, in drug delivery to target neuronal stimulation in dendrites [16,79] or for T-cell stimulation [27]. The data shown here demonstrates the feasibility of local cytoskeletal cell perturbation by induced cell shape deformations (Fig. 13.6) and a

ρ∇celltPcellt

Et=CtAt

v∇instt=dρ∇celldt

Pgradt∆ρ∇t=ρ∇bead−ρ∇cellt

0 degree<Pgradt<360 degreePgradt and Pcellt

3993 Results

FIGURE 13.5 Induced Chemotaxis by an Optically Manipulated Chemical Microsource

(A–F) An HL-60 neutrophil in the vicinity of an fMPL-releasing PGLA microsource follows the microsource motion by undergoing positive chemotaxis. The motion of the microsource was controlled via HOT (scale bar 10 mµ ), (G–H) Application of the automated cell tracking algorithm yields the cell outline [colored cell boundaries in (A–G)] and as a result also the center of mass of the cell [red dots in (A–F)]. In addition, the center of mass of the chemical microsource is depicted as black dots in (A–F), (G) the automatically tracked outline of the cell allows, for example the calculation of the cell orientation (107, 179 degrees, etc.), (H) a polar plot of the cell orientation (red) and the gradient direction (black) shows that the cell orientation follows the gradient direction. The gradient direction is defined as the direction of the vector between the centers of mass of the cell and the microsource.

Part A–F: The microscopy images adapted from Kress et al. Cell stimulation with optically manipulated

microsources. Nat Methods 2009;6:905–909 [12].

10µm

400 CHAPTER 13 Application of optical tweezers

localized lamellipodium splitting (Fig. 13.7) as reaction to perturbations with CytoD-loaded microsources (original data in Ref. [12]).

In the experiment, shown in Fig. 13.6, a bipolar stimulus was generated by two optically manipulated microsources, releasing the mycotoxin CytoD which inhibits actin polymerization [12]. The experiment was performed on an HL-60 cell which otherwise migrates randomly on the coverslip. At first, the microsources were located in front of the cell (Fig. 13.6A). Subsequently, the distance between the two beads was increased and the still migrating cell squeezed through the narrow gap between the two microsources. The lamellipodium retracted on the two sides, which were closest to the beads while the central part of the leading edge continuously ex-tended (Fig. 13.6B–F). An analysis of the cell motion with the cell shape detection algorithm shows that with decreasing distance between the centers of mass of cell and the stimulus (black dots in Fig. 13.6G) the cell velocity in -y direction (red dots in Fig. 13.6G) started to slow down significantly around t = 50 s and again later when the cell squeezed through the gap between the two microsources (starting at around t = 150 s). However, the cell orientation of roughly 270 degrees with respect to the positive x-axis did not change (Fig. 13.6H).

Since CytoD acts as an inhibitor of actin polymerization, actin-rich cell compart-ments are strongly influenced by large CytoD concentrations close to the microsources. In migrating cells, lamellipodia, thin protrusions containing quasitwodimensional actin networks, are often part of the migration machinery by acting as the leading edge of the cell [80,81]. Using optically manipulated microsources releasing CytoD, as shown in the experiment in Fig. 13.7, elegantly provides the possibility to perturb the shape and dynamics of lamellipodia and therefore also the cell polarity allow-ing, for example, the investigation of symmetry breaking and polarity establishment in cells.

In the experiment shown in Fig. 13.7, two microbeads releasing CytoD were po-sitioned close to each other in front of the center of the lamellipodium (Fig. 13.7A) of a randomly migrating HL-60 cell [12]. As a result of the locally applied CytoD, the lamellipodium retracted from the location of microsources (Fig. 13.7B–C). This led to a splitting of the lamellipodium into two parts (Fig. 13.7D–E) and the subsequent retraction of one of the lamellipodia and the expansion of the other one (Fig. 13.7F). The splitting of the lamellipodium also led to a broadening of the cell, which is rep-resented by a temporally increased cell area A(t), and a change in the cell orientation (Fig. 13.7G). With decreasing cell-bead distance, the cell orientation changed from roughly parallel to perpendicular with respect to

ρ( )∆ t , the vector between the cen-

ters of mass of the cell and of the polar stimulus (Fig. 13.7G).

3.3 THERMOTACTIC RESPONSE TO LOCAL THERMAL GRADIENTSFor the investigation of cellular responses to thermal stimuli, a similar experimen-tal approach can be used as for investigation of responses to chemical stimuli. We used optically trapped polystyrene beads with a diameter of = µd 3 mPS as highly localized heat sources. For thermal stimulation, the trapping laser was set to a total

∆ρ∇t

dPS=3µm

FIGURE 13.6 Cell Response to a Bipolar Perturbation of the Cytoskeleton

(A–F) A randomly migrating HL-60 cell experienced a bipolar perturbation of its cytoskeleton mediated by two optically trapped PLGA microbeads loaded with the mycotoxin CytoD. The cell reacted to the stimulus by retracting its lamellipodium and changing its cell shape, the red dots indicate the cellular center of mass (scale bar 10 mµ ), (G) the cell orientation did not change significantly, (H) the automated cell contour detection shows that with decreasing distance to the stimulus (black dots), the cell velocity in -y direction (red dots) slowed down significantly at around t 50s= ) and when the cell squeezed through the gap created by the two microsources (after about t 150s= ), the cell velocity in x direction did not change significantly (back squares).

Part A–F: The microscopy images adapted from Kress et al. Cell stimulation with optically manipulated

microsources. Nat Methods 2009;6:905–909 [12].

10 µm

t=50 st=150 s

FIGURE 13.7 Splitting of a Lamellipodium in Response to a Local Perturbation with CytoD

(A–F) DIC image series of a randomly migrating HL-60 cell, exposed to a localized perturbation by two CytoD-releasing microsources. The central part of the leading edge of the cell retracted from the stimulus and the lamellipodium split subsequently into two parts. The red dots indicate the cellular center of mass (scale bar 10 mµ ), (G) with decreasing distance between the beads and the cell center, the cell orientation changed from roughly vertical (90 degrees) to roughly horizontal (180 degrees). A broadening of the cell is indicated by the temporally increased cell area.

Part A–F: The microscopy images adapted from Kress et al. Cell stimulation with optically manipulated

microsources. Nat Methods 2009;6:905–909 [12].

10 µm

4034 Summary, conclusions, and outlook

laser power of 900 mW in the focal plane. With these boundary conditions, the previ-ously described temperature calibration with fluorescent dyes (Fig. 13.2) yielded a laser-induced heating of the bead of about 14 K relative to the background tempera-ture ( =T 307 KBG ) of the sample leading to a temperature increase of about 16 K/W, which is comparable to previously described values [12,57]. Cells located at a dis-tance of ρ = µ15 m from the thermal microsource and a diameter of about = µl 10 m in gradient direction experienced a temperature gradient ρ ≈ µdT d/ 0.5 K/ m between cellular front end and back end. A typical experiment investigating cellular responses to thermal stimulation is shown in Fig. 13.8.

The heat source was moved to the vicinity of an HL-60 cell (Fig. 13.8A), which started to migrate toward the heat source (Fig. 13.8B). Further movement of the heat source (black dots denote the center of mass of the chemical microsource) caused continued cell migration (center of mass of the cell depicted in red) in the direction of the source (Fig. 13.8C–F). The orientation of the cell changed continu-ously (Fig. 13.8G–H) in response to the changing direction of the thermal gradient (Fig. 13.8H). Systematic control of the thermal gradient shape or intensity can be achieved, for example, by varying the distance to the cell or by choosing different particle shapes or materials, for example, gold nanoparticles [58,59] as heat sources. Such modifications allow, for example, the systematic investigation of the cellular migration velocity as a function of the temperature and the strength of the tempera-ture gradient. However, gradient quantification strongly depends on the used fluores-cent dyes. Therefore, measuring small gradients can be difficult due to insufficient dye sensitivity.

4 SUMMARY, CONCLUSIONS, AND OUTLOOKWe have shown that optically trapped microparticles, acting as local sources of chemicals or as local heat sources, can be used to study single cell stimulation and the subsequent cellular responses. We showed that this approach can be used to induce chemotactic (Fig. 13.5) and thermotactic (Fig. 13.8) cell migration. Furthermore, optically trapped microparticles allow local cell perturbation experiments that affect the cytoskeleton in a highly localized way (Figs. 13.6 and 13.7). The large flex-ibility of optically manipulated microsources enables the application of temporally and spatially flexible stimuli to highly localized regions of cells. Complex gradient landscapes can be established by using geometries with multiple microsources (Figs. 13.6 and 13.7). In addition, contact-free positioning of the microsources by HOT is particularly useful to keep cells under physiological environmental condi-tions in a closed sample.

The chemical and thermal distribution around the microsources can be derived and measured by various techniques. The flux of chemicals from a loaded micro-source can be measured, for example, by using UV fluorescence spectroscopy or HPLC. The resulting spatial distribution of the released molecules can be calculated by solving the diffusion equation under the given experimental boundary conditions.

TBG=307 K16K/W

ρ=15µml=10µm

dT/dρ≈0.5 K/µm

FIGURE 13.8 Cell Migration in Response to a Local Stimulation With a Thermal Microsource

(A–F) DIC image series of a migrating HL-60 cell, exposed to a localized thermal stimulation. The polystyrene microsource with a diameter of d 3PS = µm was moved in a clockwise direction by HOT (black dots denote the center of mass of the bead) and was followed by the cell. The red dots depict the cellular center of mass. Gaps in the center of mass trajectory (between t 122 s= and t 168 s= and between t 273 s= and t 291 s= ) result from failure of the detection algorithm to separate individual cells when two cells touch (scale bar 10 mµ ), (G) the cell orientation followed the gradient direction by spanning an angular range of φ∆ >180 degree, (H) the polar plot of the cell orientation (red) and the gradient direction (black) shows that the cell orientation followed the gradient direction. The gradient direction was defined as the direction of the vector between the centers of mass of the cell and the microsource.

dPS=3

t=122st=168st=273st=291s10µm

∆φ>180 degree

4054 Summary, conclusions, and outlook

In the case of thermal microsources, the temperature profile around the particles can be obtained by using temperature-dependent fluorescent dyes.

Optical tweezers allow the trapping of microparticles made from a wide range of materials [12,82,83]. This gives the possibility to optimize the materials for specific experimental needs in studies involving chemical or thermal cell stimulation. The biochemical stimulation experiments presented here were performed using PGLA microspheres, which were originally designed for controlled drug release over long time periods of up to several weeks [49]. In contrast, microsources made of colloido-somes [84] or mesoporous silica nanoparticles [85] could offer faster release rates and therefore increased concentrations of the released chemical. For optically heated microsources stimulating thermotactic cell response, the achievable temperatures de-pend not only on the used laser power but also on the absorption coefficient of the particle material and can therefore be optimized by the choice of the most suitable material. Optical trapping and heating of plasmonic nanostructures, such as metallic nanoparticles, for instance strongly depends on the shape of the structure [58,59,86], therefore enabling the generation of entirely different heat gradients (short range and very steep near the particle), which might affect cells differently.

HOT allow controlling the number of microsources as well as their spatial dis-tribution [12,37]. Furthermore, the duration of the stimuli can be controlled by the temporal flexibility of HOT. These tuning options and the inherent flexibility to use different types of stimuli in the same experiment can be highly important for studies on cells that are able to respond to various potentially conflicting stimuli. For exam-ple, in the case of immune cells that are able to undergo chemotaxis and thermotaxis, optically manipulated microsources can be used to apply simultaneously a chemical and a thermal gradient in different directions to investigate whether, for example, one of the two stimuli dominates over the other and determines the cell response. Therefore, the earlier-mentioned flexibility of optically trapped microsources can be used, for example, to experimentally dissect purely thermotactically and purely biochemically induced effects and to gain a better understanding of their strength and possible interplay, for example, in cancer metastasis [23,87].

Automated cell shape detection provides a highly defined framework for data analysis in assays targeting cell motility and morphology. Compared to manual de-tection methods it has been shown to yield superior precision and reproducibility [75,88]. During the experiment, it gives rise to the possibility to track the cell shape with a high frame rate and can be a time saver for the experimentalist during post-processing, allowing high data throughput. Various parameters, such as the area, the grade of elongation, the orientation, the centroid, and the velocity of the centroid can be extracted and analyzed with regard to the local cell stimulation.

The algorithm used here for the segmentation of cells in DIC images is based on local contrast detection. We have shown that the calculation of local grayscale extrema in combination with simple morphological operations is sufficient to seg-ment eukaryotic cells in DIC images. Since the calculation of minima and maxima is very efficient with modern CPUs, the algorithm is suitable for live tracking with a framerate between 1 and 10 Hz on 1000 × 1000 pixel images, depending on the

406 CHAPTER 13 Application of optical tweezers

number of cells and the quality of the input images. In contrast to image reconstruc-tion techniques, it does not require additional information about the setup, such as the orientation of the DIC shear axis, which can differ between multiple datasets. The algorithm is therefore easy to implement and use.

Consequently, the possible combination of the presented techniques of optically manipulated microsources and automated cell shape detection with other experimen-tal methods for life-cell measurements, such as elastic substrates of different stiffness, microfluidic devices, fluorescence microscopy, or the other various light robotics tools described in this book will provide a powerful work station for investigating complex cellular processes.

ACKNOWLEDGMENTSWe would like to thank Orion Weiner (UC San Francisco) for providing HL-60 cells and Stefan Conrad (University of Bayreuth) for helpful discussions. Furthermore we would like to acknowledge support from the German Research Foundation (Deutsche Forschungsgemein-schaft, KR 3524/4-1), the Elite Network of Bavaria (ENB) and the German Academic Scholar-ship Foundation (Studienstiftung).

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CHAPTER

Light Robotics Structure-mediated Nanobiophotonics. http://dx.doi.org/10.1016/B978-0-7020-7096-9.00014-8Copyright © 2017 Elsevier Ltd. All rights reserved.

Cornelia Denz, Álvaro Barroso PeñaInstitute of Applied Physics, University of Muenster, Muenster, Germany

CHAPTER OUTLINE

1 Introduction ...................................................................................................... 4112 Nanomotors ...................................................................................................... 412

2.1 Bio-(inspired) Microscopic Motors ...................................................4132.2 Directional Control Methods ............................................................4152.3 Speed Regulation and Temporal Motion control .................................416

3 Bacterial Microsystems ..................................................................................... 4173.1 Bacterial-Powered Microfluidics Pumps ............................................4173.2 Bacterial Microrobots .....................................................................418

4 Bacterial Adhesion and Patterning ..................................................................... 4194.1 Theory of Bacterial Adhesion ...........................................................4194.2 The Role of Cell Surface Structure Mediating Bacterial Cell Adhesion ......4204.3 Control of Bacterial Adhesion and Patterning ....................................422

5 Optical Manipulation of Bacteria ........................................................................ 4235.1 Optical Trapping of Microscopic Particles .........................................4245.2 Advanced Optical Control ...............................................................4255.3 Photodamage on Biological Samples During Optical Trapping .............427

6 Functional Optically Assembled Systems Based on Biomolecular Motors .............. 4286.1 Bacteria-Based Microfluidic Mixers ..................................................4296.2 Bacterial Microrobots .....................................................................430

7 Conclusions ...................................................................................................... 432References .............................................................................................................. 432

1 INTRODUCTIONThe manipulation and actuation of biological and engineered soft materials in the micro-and nanoscale are main requirements toward the construction and use of microscopic devices with specifically defined functionalities. On the one hand, optical forces allow a sophisticated control of microscopic objects with

Controlling autonomous nanobiorobots by optical micromanipulation

14

412 CHAPTER 14 Controlling autonomous nanobiorobots

high spatiotemporal resolution. For example, needle-shaped diatoms have been optically trapped and steered for cell surface probing and imaging [1], while a large number of light-driven complex-shaped microrotors have been applied for defined microflow induction [2–6]. Furthermore, two-photon polymerized mi-crotools and motors have been driven by light [7–9], and used as light-deflecting guides for targeted-light delivery [10,11]. Indeed, the various chapters in this book present a rich variety of promising possibilities in light robotics. On the other hand, the exploitation of biomolecular motors as mechanical actuators for the implementation of microsystems and microrobots have opened new exciting possibilities in the field of nanoengineering and biomedicine. In contrast to the smallest man-made actuators which are tens of micrometers in size [12], biomo-tors are much smaller, ranging from the micro to the nanometer range. Particular-ly, biomotors that propel swimming cells allow sophisticated motion via a variety of complex mechanisms.

Biomotors can play an essential role in creating autonomous functional robots at the nano- or microscale. They can provide viable solutions especially in working environments that are inaccessible to high-quality trapping beams. A challenging task for a reasonable utilization of such biological robotic motors in a general and efficient cooperative behavior is to attach, that is, adhere or tether a single or a mul-titude of individual biological cells to a specific surface or microparticle in a defined way. Optical actuation methods can interface with biomotor-based approaches in this respect. In this chapter, we outline the role of optical manipulation methods as a general solution to control micropatterning and microassembly of bacteria- based biohybrid nano- and microrobotic devices. We discuss the application of complex light landscapes as three-dimensional traps for achieving full control of a multitude of bacteria featuring a variety of shapes, and we give a comprehensive overview of functional optically assembled biohybrid systems that exploit the unique features of flagellar molecular motors for powering microsystems.

2 NANOMOTORSThe design and fabrication of efficient, robust, and self-powered microsystems and microrobots is one of the most exciting challenges facing nanotechnology. One of the first challenges to address is certainly the use and control of nanoscale motors for the precise actuation and propulsion of these microsystems and microrobots. Steer-ing and navigating the nanomotor of these microsystems is essential for addressing complex operations in fluidic media, such as drug delivery, but also for nanoscale assembly or patterning. In the first of the following Sections (2.1), an overview of different types of autonomous bio-(inspired) nanomotors is presented with special focus on bacterial flagellar motors. Then, available methods for directional control of biomolecular motors are described and categorized as structural-based or taxis-based (Section 2.2). Finally, temporal control of the motion and on-off switch mechanisms are discussed in Section (2.3).

4132 Nanomotors

2.1 BIO-(INSPIRED) MICROSCOPIC MOTORSActuation of microscopic objects is an extremely challenging task, particularly for microsystems and microrobots designed to operate in fluidic media due to the combi-nation of Brownian motion and low Reynolds numbers (where viscosity dominates). Accordingly, navigation principles in the macroscale world are not applicable for na-noscale propulsion. Indeed, during billions of years of evolution, living cells have optimized a large complex system of protein-based machines that allow single cells to perform complicated tasks in this regime, such as cell division, self-propulsion, and environment sensing, to mention a few. In addition, these biomolecular machines directly convert chemical energy from the surrounding medium to mechanical energy with high efficiency. This autonomous actuation enables the reduction of the size of the machine by overcoming the challenge of electrical energy storage and genera-tion that make miniaturization of self-powered untethered artificial microsystems and microrobots so difficult. Therefore, it is not surprising that in the last decade biomolec-ular motors have been used to inspire the design and operation of artificial microscopic motors, but also used themselves as self-powered actuators in microsystems.

The first bioinspired artificial microswimmers were developed starting with the controlled swimming motion of an artificial flexible flagellum made from a linear chain of colloidal magnetic particles linked by DNA and attached to a red blood cell [13]. This led to the development of artificial magnetic nanostructured propellers [14], and micrometer-scale artificial bacterial flagella [3]. These microscopic helical propellers actuated by external sources of power have been successfully used for manipulation of microobjects [15] and more recently for transporting sperm cells with motion deficien-cies [16]. Also inspired by biological motors, synthetic molecular motors (and par-ticularly chemically powered catalytic nanomotors) represent an alternative approach toward the elimination of the external source of power for actuation [17]. In compari-son to biological machines, synthetic nanomotors might offer considerable advantages in the development of complex nanomachinery since their customized design can enable them to be used for specific tasks and in a more diverse range of conditions.

However, in a more practical point-of-view and considering the difficulty of reproducing artificially the complexity of biomolecular motors, a trending strategy is to use these molecular machines for actuation purposes instead of mimicking them artificially. Biomolecular motors are complexes of two or more proteins that convert chemical energy, most commonly adenosine triphosphate (ATP), into directed mo-tion with a remarkable efficiency. Some of the most exploited motors for nanotech-nological applications are the cytoskeletal motor proteins of eukaryotic cells, and in particular, the microtubule-based kinesin and the actin-based myosin motors [18]. These motors move linearly in either a processive or nonprocessive fashion along the corresponding cytoskeletal filaments, which act as motor “tracks,” and are in-volved in almost every aspect of controlled motion and force generation within cells, for example, intracellular transport of materials [19–21]. Thus, they can be applied for the transport, sorting, self-assembly, and detection of nanosized cargo, such as nanoparticles or DNA, as well as for biosensing and diagnostics [22].

414 CHAPTER 14 Controlling autonomous nanobiorobots

Another well-known and widely exploited biomolecular motor is the rotary F1-ATPase molecular motor that is used by flagellated bacteria, such as Escherichia coli or Bacillus subtilis as a propulsion mechanism [23]. These rotary motors are embedded in the cell wall and are connected to stiff helical protein filaments called flagella. For self-propulsion, these bacteria use the free energy stored in transmem-brane gradients allowing a controlled ion flux that generates a torque for rotating the flagella [24]. When the filaments rotate in synchrony they form a coherent-ly rotating bundle that propels the cell forward (Fig. 14.1A,D). This “run” mode alternates with the “tumble” mode when one or more flagella motors reverse their rotation (Fig. 14.1 B,E), which induces a reorientation of the bacterium before it starts swimming again (Fig. 14.1C,F), resulting finally in a three-dimensional random walk at neutral, gradient-free conditions [25] (Fig. 14.2A). Interestingly, F1-ATPase was reported to convert chemical energy into mechanical energy with nearly 100% efficiency [26]. The first in vitro implementations using this rotary biomolecular motor were demonstrated by fixing the stator to a nanostructured sur-face and exploiting the movement of the rotor for rotation of fluorescent micro-spheres [27], as well as for actuation of a fabricated inorganic nanopropellers [28].

FIGURE 14.1

(A–C) Schematic of self-propulsion mechanisms of flagellated bacteria like B. subtilis or E. coli, and (D–F) corresponding experimental fluorescence microscopy images. (A, D) When all flagella rotate counter-clockwise they can bundle and enable the bacterium to swim efficiently. With a certain probability one or more flagella occasionally rotate in the contrary direction, causing the flagella to unbundle (B, E). This induces a random reorientation by means of tumbling of the bacterium before it starts swimming again (C, F).

Fluorescence microscopy images were kindly provided by Prof. Berenike Maier and Jan Ribbe both from

University of Cologne.

4152 Nanomotors

Moreover, due to the small size of flagellated bacteria between 1 and 5 µm and their high efficient mechanism for motion in the low Reynolds number regime, they have been also proposed as self-powered actuators for microfluidic applications, transport of microparticles, and powering microsystems [29], as will be described in detail in the following sections.

2.2 DIRECTIONAL CONTROL METHODSRegulating the movement of biological motors is a fundamental requirement for the propulsion control of biohybrid microsystems. For example, such directional control might be desirable to pick up a cargo at a certain position, move it along a pre-defined path, and then release it. Methods for such spatial control of biomotors can be categorized into two main strategies: structural-based and taxis-based approaches.

Under structural-based approaches are included those assays by which the spatial control is achieved by constraining the trajectory paths of the biomolecular motors. A straightforward case is the so-called stepping assay, in which the cytoskeletal fila-ments are laid out on a surface where they form tracks for the motors to move along in a linear trajectory [30]. The structural polarity of these filaments featuring a plus and a minus end enables a unidirectional trajectory of these motors, and accordingly of any organelle or nano- and microparticle attached to them, along their tracks [31]. In order to control the direction of motion in stepping assays, the filament arrays on the surface have been oriented in a predefined direction by binding specific filament ends to a surface and using an additional fluid flow to align the filaments along the surface [32]. In case the motors are immobilized on a surface and the filaments glide over them, the so called gliding assay, a combination of chemical and topographic patterning (acting as obstacles) can provide efficient guiding and spatial control of the filament movement in particular paths [30].

When using flagellated bacteria as self-powered actuators, directional control employing geometrically-selected environments has been achieved with fluidic microchannels and/or 3D microfiber structures, by which the swimming paths of

FIGURE 14.2

Schematic of (A) random and (B) directed movement of a single bacterium when it is immersed in a medium with absence and presence of a gradient of nutrients, respectively.

416 CHAPTER 14 Controlling autonomous nanobiorobots

the bacteria were restricted for conducting microtransport of bacteria [29]. Defined geometries and solid planar surfaces can also be exploited to influence the motion be-havior of flagellated bacteria [33]. Other environmental conditions can also influence the swimming direction of bacteria. For instance, porous media has an effect on the random walk for bacterial migration [34].

Another possibility for directional control is the utilization of taxes-based approaches, which mainly apply for the movement of a large number of motile cells. These taxes, investigated with light robotics in Chapter 13, are considered either positive toward the source or negative away from it and can enable steering control of a single or several bacteria to follow precisely a predefined path or trajectory [29]. On the one hand, there are taxes that trigger bacterial stimulus-response mecha-nisms through environmental sensing. These sensory-based reactions influence the tumbling frequency in such a way that they can be guided to specific locations. These taxes include chemotaxis (directed motion toward a chemical attractant or a nutrient-rich zone and away from toxins) [35], as depicted in Fig. 14.2B, aerotaxis (motion of a microorganism toward or away from air or oxygen) [36], and phototaxis (movement toward or away from stimulus of light) [37]. On the other hand, there are taxes that can be controlled by means of off-board fields. These include deterministic taxes modes as galvanotaxis (motion directed toward a cathode or anode, depending on swimming cell) [38] and magnetotaxis (induced movement by magnetic fields), to name some examples. In addition, electric fields can induce the actuation of bacterial microrobots by means of electrophoretic forces [39].

2.3 SPEED REGULATION AND TEMPORAL MOTION CONTROLIn addition to directional motion control, the precise temporal motion control of nanoscale biomolecular motors, including fine regulation of the motor speed and cyclic “on/off” activation also known as stop-resume, is an essential requirement for the performance of some specific tasks of biohybrid microsystems. In this re-spect, various parameters affecting biomolecular motors activity can be used to regulate their speed, including chemical stimuli, thermal or light-induced speed modulation [29].

At the single-molecular level, rotation of F1-ATPase based nanopropellers has been initiated using ATP which can be inhibited by sodium azide [28]. In addi-tion, binding of heavy metals to the rotor of the flagellar motor impairs its motion instantaneously, and thus, rapid stop/resume motion of the flagellar motor of motile bacteria was accomplished by adding in the bacterial media copper ions and the ethylenediaminetetraacetic acid (EDTA) chelating agent, respectively [40]. It was also discovered a while ago that the temperature influences motility and that the chemotaxis behavior of bacteria likewise depend on the temperature. In another re-port, thermal on/off switching of the rotary motor F1-ATPase was addressed using thermally responsive nanomeshes.

Moreover, light-modulated motion has also been described for controlling both the movement of linear and rotary biomolecular motors [41,42]. This control was

4173 Bacterial microsystems

accomplished by exploiting a UV-induced release of caged ATP combined with enzymatic ATP degradation by hexokinase to turn the molecular shuttles “on” and “off” sequentially. For the rotary motor, UV-induced stop/resume was proposed to control the flagellar rotational motion and hence affecting the motility of the bacteria.

Other environmental procedures are also possible, such as the addition of oxy-gen and arginine that can play a role on bacterial motility. Additionally, structural and environmental approaches, such as the bacterium proximity to solid surfaces or the increase of the viscosity of the extracellular medium can reduce bacterial swimming speed.

3 BACTERIAL MICROSYSTEMSThe directed and temporal control motion of bio-(inspired) molecular motors has been extensively exploited in the last years for the actuation and propulsion of syn-thetic microsystems. Particularly, nanomotors that rotate the flagella filaments of prokaryotic cells through the collective motion of several or few bacteria have been applied for the actuation of two types of microsystems: microfluidic flow genera-tion either by the flagellar motion or by bacterial-driven microdevices and transport in aqueous media of inanimate synthetic micro- and nanoparticles. In this section, we review such bacterial-powered microfluidic pumps for the first actuation example (Section 3.1), and discuss implementations for the case of bacterial microrobots in the second part (Section 3.2).

3.1 BACTERIAL-POWERED MICROFLUIDICS PUMPSE. coli and other motile bacteria are propelled through water by several helical fla-gella, which are in turn rotated by motors embedded at random points on the cell wall. Depending on the handedness and rotation sense, the motion of the flagella induces a flow field that causes them to wrap around each other and form a bundle. Full-scale flagella are around 7 µm in length, 12 nm in diameter, and turn at a rate of 100 Hz [25]. Experimental models mimicking this environment have been created in order to investigate mechanics underlying this process [43,44].

Originally, experimental studies taking advantage of the swimming capability of bacterial cells microsystems consisted of a monolayer of flagellated bacteria attached to a solid–liquid interface. This so-called bacterial carpet, which formed a densely packed bacteria monolayer with their flagella rotating freely in the me-dia, was demonstrated to pump liquid in a microfluidic channel [45,46] and to enhance mixing in microfluidic systems [47], as it is depicted in Fig. 14.3. Control of the overall mixing and pumping properties of these bacterial pumps can be regulated by the concentration of glucose, the system temperature, the microflu-idic channel width, and the chemotactic swimming characteristics of flagellated bacteria [48,49].

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In a step further, bacterial motility was used for actuation of artificial rotating-objects [50], achieving subsequently microfluidic flow motion in the sample media, and the transport of particles by means of asymmetric submillimeter gears activated upon random movement of bacteria [51]. Such remarkable experiments stimulated research efforts to model these asymmetric biohybrid micromotors in chaotic bacte-rial baths [52,53].

3.2 BACTERIAL MICROROBOTSLinking biomolecular motors of cells or whole self-propelling cells with inorganic micro- and nanoscale particles, results in the so-called biohybrid microrobots or living micromachines. These have key advantages for the transport of micro- and nanoscale objects in aqueous solutions. Since they convert chemical energy from the surrounding medium, they overcome the challenge for miniaturization of the en-ergy power source. The reduced size of these microrobots allows them to be used as transport vehicles to otherwise inaccessible places. In combination with micro- and nanocontainers as carriers of drug molecules or as agents for sensing and imaging applications, these biohybrid microrobots have been envisioned as futuristic tools for on-board diagnosis and therapy [54]. The first conceptual model of a biohybrid microrobot prototype involved members of the Paramecium genus, a group of uni-cellular cilia-propelled protozoa, thus much larger than a bacterium [55]. Later, sin-gle bacterial cells were proposed as self-propelled microcarriers by attaching them to artificial objects of few micrometers to tens of nanometers in size.

For directional control of the transported particle powered by flagellated bacteria, various bacterial taxes have been considered. For example, bacterial chemotaxis has

FIGURE 14.3

(A) Schematic of the bacterial mixing system using a Y-junction microchannel and a bacterial carpet covering the top and bottom of the channel. (B–C) Typical intensity distributions in the Y-junction microchannel, when (B) the walls of the microchannel are free of bacteria, and (C) the channel is coated with an active bacterial carpet.

Adapted from Kim MJ, Breuer KS. Use of bacterial carpets to enhance mixing in microfluidic systems.

J Fluids Eng 2007;129: 319 [47].

4194 Bacterial adhesion and patterning

been employed for propelling microbeads [56] and sorting micro- and nanoparticles [57,58], phototaxis has been implemented for controlling microfabricated structures [37], and magnetotactic bacteria have been used for the transport of samples in mi-crofluidic microsystems [29]. In analogy to the use of asymmetric microscopic gears in bacterial baths, the introduction of an asymmetric shape to the transported particle constraints the directionality of the bacterial propelled system [59,60]. In addition, a targeted and spontaneous transport of spherical particles can be achieved by immers-ing them in an active bacterial bath within a designed three-dimensional microstruc-ture with slight asymmetries [61].

Various types of microobjects have been employed as the abiotic part of these living micromachines. For instance, several bacteria have been attached to frag-ments of polydimethylsiloxane, to polystyrene beads, and to PDMS microcylinders [45,62]. With the idea of performing designed biomedical tasks, recent attention has shifted to strongly functional objects capable of carrying and delivering therapeutic and diagnostic agents. In this respect, porous materials are highly interesting since they can accommodate a variety of guest molecules. For instance, zeolites L crystals, which are framework aluminosilicates with inner one-dimensional nanochannels, can be employed as nontoxic nanotransporters [63]. Furthermore, they can be func-tionalized on their surface and channel entrances in order to enable more complex assemblies than conventional microspheres. Zeolite L crystals thus serve as a very versatile building block to form biohybrid microrobots. Whereas the interior nano-channels of zeolite-L crystals can be loaded with drug molecules or luminescent dyes for imaging and labeling biological cells [64], the outer surface can be chemically modified to allow assembly and targeting of the bacteria, with subsequent transport of the zeolite L nanocontainer by the attached motile bacterium [65].

4 BACTERIAL ADHESION AND PATTERNINGThe actuation, that is, manipulation and transport, of artificial microparticles is typi-cally realized by attaching the bacteria to a synthetic microstructure. Thus, bacterial adhesion is a crucial step toward the fabrication of biohybrid microsystems. In addi-tion, microbial adhesion is also relevant in other biological processes as in bacterial colonization and biofilm formation. Thus, the mechanisms of adhesion to abiotic surfaces must be elucidated for an effective fabrication of autonomous bacterial-based microrobots. Section 4.1 presents a brief description of the theory of bacterial adhesion. Then, the role of the bacterial cell surface composition is discussed for a more complete description of the complex adhesion process (Section 4.2). Finally, technologies for controlling microbial adhesion are described (Section 4.3).

4.1 THEORY OF BACTERIAL ADHESIONBacteria are about 0.5–2 µm in size and typically present, as natural surfaces, a net negative charge at their surface [66]. Hence, counter ions in the aqueous bacterial

420 CHAPTER 14 Controlling autonomous nanobiorobots

media are attracted to them, forming an electric double layer. In a first approximation, interactions of bacteria with other organisms and abiotic surfaces can be analyzed in the frame of soft matter physics and chemistry on the basis of colloidal interactions and on the interplay of a colloidal particle with a surface [67]. These are described by the DLVO theory established by Derjaguin, Landau, Verwey, and Overbeek, as the summation of their van der Waals and Coulomb interactions [68,69].

According to this theory, the interaction energy has a primary deep minimum in the vicinity of the surface at high ionic strength of the media (Fig. 14.4A), leading to irreversible bacterial adhesion when the bacteria come close to the surface by either swimming or Brownian motion. At medium ionic strengths, an energy barrier between the primary minimum and a secondary shallow energy minimum appears at typically several nanometers from the surface (Fig. 14.4B). In this case, bacterial adhesion is produced as a two-step or one-step process (Figs. 14.4D and 14.4F, respectively) [70]. In the first case, a bacterial cell comes first to the secondary minimum by its motility or Brownian motion and adheres to the surface weakly, often reversibly. In a second step, irreversible primary minimum adhesion is accomplished by means of nanofibers of the cell which can pierce the energy barrier due to their small radii [66]. In the one-step adhesion, adhesion can occur by direct contact of long nanofibers to the par-ticle surface. Fig. 14.4E,G–H show respectively exemplary experimental images of a single bacterium attached to a microparticle by small and long cell appendages.

Finally, at low ionic strengths, the repulsive energy barrier increases due to shielding of the surface charges by which the ionic action in the electrical double layers decreases (Fig. 14.4C). As a result, bacterial cells cannot surmount this barrier and cell adhesion does not occur. Such relation between decreasing bacterial adhe-sion and decreasing ionic strength has been investigated in numerous experimental studies, showing a clear consistency with DLVO theory.

An alternative description of bacterial adhesion is based on a thermodynamic ap-proach which takes into account the surface free energies of the interacting surfaces, that is, solid–microorganism, solid–liquid, and microorganism–liquid. Although, the ther-modynamic approach cannot be applied to the adhesion in phase one, that is, at the sec-ondary energy minimum where a new cell–substratum interface cannot be formed, the thermodynamic approach explains a common observation: bacteria with a hydrophobic cell surface prefer hydrophobic material surfaces, while those with a hydrophilic cell surface prefer hydrophilic surfaces [71,72]. The extended DLVO theory that includes this effect in the description of bacterial adhesion is reviewed in more detail in [66].

4.2 THE ROLE OF CELL SURFACE STRUCTURE MEDIATING BACTERIAL CELL ADHESIONActual bacterial adhesion is an extremely complex process and frequently deviates from the adhesion models described before. On the one hand, solid materials adsorb various organic and inorganic matter to their surfaces forming layers with physico-chemical properties quite different from the original bare surfaces. On the other hand, unlike simple colloidal particles, bacterial surfaces are structurally and chemically

4214 Bacterial adhesion and patterning

heterogeneous, featuring various kinds of proteins appendages embedded in the cell membrane, which directly affects microbial adhesion to solid surfaces. Importantly, these cell appendages bridge cells to the substratum, as already described in the sec-ond step of bacterial adhesion in neutral conditions, and thus have an important role in the specific and nonspecific cell adhesion to biotic and abiotic surfaces.

There are mainly two types of cell appendages involved in bacterial adhesion and biofilm formation: proteinous nanofibers and polysaccharide chains [66]. Among polysaccharides chains, lipopolysaccharides (LPS) are the most relevant for bac-terial adhesion. In Gram-negative bacteria, LPS are contained in the lipid bilayer outer membrane (OM) with significant variations in the coverage density and local

FIGURE 14.4

(A–C) Dependence on ionic strength of the total interaction energy between a bacterium and a surface. (D) Schematic of the two-step adhesion model, and (E) an exemplary image of a single B. subtilis attached in this way to a zeolite L nanocontainer. (F) Schematic of the one-step adhesion process mediated by a long nanofiber and (G–H) an example of a motile B. subtilis attached by a flagellum to two zeolite L nanocontainers. Adhesion of the bacterium to the particles is demonstrated by the propulsion of the zeolite by the swimming bacterium.

Schematic images adapted from Hori K, Matsumoto S. Bacterial adhesion: from mechanism to control.

Biochem Eng J 2010;48:424–434 [66].

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distribution. Lipopolysaccharides are important at the initial step of bacterial adhe-sion to surfaces since they create an energy barrier, and thus they hinder close contact of cells with surfaces. Other relevant polysaccharide chains, as, for example, exo-polymeric substances (EPS), are important for the development of biofilm structure rather than for cell adhesion. The reason being that a reduction of EPS blocks biofilm development, but does not prevent the first step of cell adhesion, while an increased amount of EPS leads to thick biofilms [66].

In addition to these polysaccharide chains, other proteinous cell appendages also mediate in cell adhesion to abiotic surfaces. They are involved in biofilm forma-tion, as well as in pathogenic infection by binding to biomolecules on host cells and/or extracellular matrices (ECMs). These cell appendages are present in many kinds of bacteria and their diameter range from several to tens of nanometers, while their lengths range from hundreds to thousands of nanometers. In particular, pili are a well-known bacterial hair-like nanofiber that function as adhesins, that is, cell- surface appendages that facilitate bacterial adhesion. Typically, a bacterial cell has more than one type of pilus, which vary in function, molecular structure, localiza-tion on the cell surface and mechanisms of secretion and assembly. Other nanofibers playing a role in bacterial adhesion, to mention a couple, are the peritrichate “hamus” nanofiber of the archaea cells and the adhesive “stalk” nanofiber of the bacterium Caulobacter crescentus. The last one mediates the strong adhesion of the stalked cells to surfaces with around 70 N/mm2, which is the strongest adhesion strength ever measured for a biological adhesive. A more detailed review of bacterial nanofibers and polysaccharides chains can be found in [66].

As described earlier, these bacterial nanofibers might pass through the energy barrier at normal ionic conditions in order to tether the cell body to the surface, but they also make cell adhesion to deviate from the behavior predicted by the DLVO theory. In this respect, a parameter that estimates the height of the energy barrier is the Zeta potential, which is typically calculated from the electrophoretic mobility [73]. Interestingly, it has been found that calculation of the Zeta potential using the Smoluchowski equation for rigid particles without polymer surfaces [74], results in a nonzero value with increasing ionic strength which differs from experimental observations. However, accurate surface potentials of actual bacterial cells can be calculated by applying a more precise model, the Ohshima model, which includes particles with a soft polymer surface layer [75,76]. As a result, the actual surface potential of bacterial cells is much smaller than the Zeta potential obtained from the Smoluchowski equation. Moreover, the energy barrier actually disappears or is sufficiently low for bacterial cells to surmount it even at low ionic strengths, where, by applying the Zeta potential to the DLVO theory, a high-energy barrier had been thought to prevent direct interaction between a cell and a surface [77].

4.3 CONTROL OF BACTERIAL ADHESION AND PATTERNINGBacterial adhesion can be produced through various mechanisms, including physicochemical approaches, ionic attachments, hydrogen bonds, the use of

4235 Optical manipulation of bacteria

antigens, the exploitation of hydrophobic properties, and electrostatic parameters [29,66,67,70]. Interestingly, it has been shown that bacterial adhesion is enhanced when surfaces are positively charged, but subsequent biofilm formation is slower. This indicates that a positively charged surface adversely affects biofilm growth. It was found that approximately 80% of E. coli cells and 60% of B. subtilis cells adhering to highly positively charged surfaces were inactivated after a contact time of 8 h [78].

Reversible bacterial adhesion including cells on cells or cells with polystyrene surfaces using photochemistry has also been demonstrated. Other approaches for controlling bacterial adhesion have also been reported based on electroassisted methods that utilize, for example, the electrorepulsive interaction between bacteria and cathodic surfaces with a negative surface charge [79].

Furthermore, successful bacterial adhesion have been demonstrated on many different materials including, among other, polymeric surfaces like polydimethylsi-loxane [45], polymer brushes [80], zeolite L/polymer hybrids [81], and polystyrene [82], SU-8 microstructures [37], and glass [83].

However, established methods for the formation of biohybrid microsystems rely typically on an uncontrolled attachment of the biological specimen to the abiotic surface which is far away from desired controlled and defined attachment, especially in a patterned way to create coherent collective action. Considerable progress has been made to tackle this problem by developing effective methods for patterning and chemical modification of surfaces and particles. For example, surface pattern-ing has been employed using polymer film templates [84] and by soft lithography [85]. Additionally, promotion or reduction of microbial adhesion can be addressed using topographical features on surfaces, which provide more or less available bind-ing sites. A successful example was demonstrated using nanofiber-textured model surfaces [86]. In the case of microscopic particles, site-specific functionalization has been proposed so that the biological specimen attaches only where the surface has been chemically modified [65,87,88].

Besides these techniques, optics has been recently come into play to enable or enhance adhesion. These optical methods will be explained in the following section.

5 OPTICAL MANIPULATION OF BACTERIAA challenging task for the efficient assembly and formation of biohybrid systems based on biomolecular motors is tethering or attaching a single or a multitude of individual nanobiomotors to a specific surface or microparticle in a defined and con-trolled way. As described in Section 4.3, conventional fabrication and bonding tech-niques for the assembly into biomolecular-based microdevices rely on site-selective functionalization of the abiotic object surface. In this way, the biological specimen attach only where the surface has been modified. However, micropatterning and microassembly processes require a relatively high effort, suitable material, precise biochemical know-how, and involved equipment.

424 CHAPTER 14 Controlling autonomous nanobiorobots

Moreover, they offer only a limited spatiotemporal control of the bacterial adhe-sion and lack fine control over individual bacteria despite the fact that in the context of microrobotic fabrication, a precise and robust bacterial positioning and patterning on surfaces is of utmost importance. Micromanipulation techniques that rely on non-contact approaches offer tremendous advantages with respect to particle control, minimized damage to the bacterial specimen, and reconfiguration of bacterial as-semblies on particles and surfaces.

Actual techniques allowing an enhanced spatial and temporal control of cell adhe-sion are based on microfluidics [89], electric currents [90], dielectrophoresis [91,92], and especially optical forces interacting with the cell as described in more detail in this chapter. In the following paragraphs, we briefly comment about optical tech-niques for manipulation and investigation of biomolecular motors (Section 5.1), the use of advanced optical trapping for full three-dimensional orientation control and bacterial arrangement (Section 5.2), and finally we discuss the photodamage induced to the biological specimen during optical trapping and manipulation (Section 5.3).

5.1 OPTICAL TRAPPING OF MICROSCOPIC PARTICLESAmong the contactless methods available for manipulation of micro and na-noscale particles, laser light-based techniques have been demonstrated to enable unprecedented versatile control of transparent particles. In particular, one of the most commonly used trapping approaches nowadays are optical tweezers, which enable manipulation of particles near the focus spot of a tightly focused laser beam [93,94]. Once a particle is trapped, the particle can be moved by mechanically steering the trapping beam with scanning mirrors or by translating the microscope sample rela-tively to the laser beam. Chapter 1 discusses the basic principles of optical trapping systems further.

Optical tweezers have been widely applied, not only to trap and manipulate inani-mate particles [95], with different shapes and sizes ranging from several micrometers down to the nanometer scale, but also to study single-molecule studies in the biology field, for example, analysis of molecular motors or nucleic acid molecules [96]. Opti-cal tweezers have also been employed to trap and investigate single living cells and organelles within cells [97]. In particular, by trapping motile bacteria with optical tweezers, valuable access to the biophysical properties of flagellar nanomotors can be obtained. For instance, long-term characterization of the run-tumble behavior of individual E. coli cells was demonstrated using a system which combined dual beam optical tweezers, bright-field and fluorescence microscopy [98]. In addition, interac-tions of a silica particle and a bacterium, using dual beam optical tweezers, provide direct monitoring of the flow generated by bacterial rotation [99]. Differences in bacterial motility between mutants and wild-type strains of a same bacterial cells and the toxic effect of an antimicrobial agent on single bacterial cells were moni-tored and determined using single-beam optical tweezers [100,101]. With respect to spatiotemporal bacterial adhesion control, simple optical tweezers have been used for a multitude of different approaches: for assisting the positioning of bacteria at

4255 Optical manipulation of bacteria

surfaces in a predetermined area [102], for investigating the influence of specific nanodomains at the pole of rod-shaped bacteria (bacterial polarity), for adhesion to glass [103], for measuring forces between bacteria and functionalized coated mi-crospheres [104], and for studying the role of bacterial nanofibers [105] in bacterial adhesion processes.

When the biological object is too tiny to be trapped and observed which typically is connected to a size smaller than the wavelength of the light used to trap the object, for example, cytoskeletal motor proteins or bacterial nanofibers, or when photo dam-age by direct laser exposure is limiting its use, indirect optical manipulation might be an attractive alternative [106]. For this purpose, dielectric particles (made of latex, polystyrene, silica, etc.) are optically-trapped and attached to the biological object so that they can serve as handles for manipulation and deformation of the small and sensitive biological object, realizing a delicate and careful means of sensing forces in biomedical environment. For instance, indirect manipulation allow stretching of nucleic acids, for example, DNA and RNA, investigating their biomechanical prop-erties [97]. Indirect micromanipulation can also be used to trap nanoparticles driven by cytoskeleton biomolecular motors in order to determine the forces applied by the motor to move the cargo. Moreover, indirect manipulation by optical tweezers allows studying bacterial adhesion not only on the single cell level, but also at the single adhesion organelle and at the single adhesin-receptor interaction level, which pro-vides key information about given adhesin-receptor interactions involved in bacterial adhesion [107].

5.2 ADVANCED OPTICAL CONTROLWhile the investigation of a single bacterium with conventional optical tweezers gives insight into some of its properties and features, access to the orientation con-trol of asymmetric (e.g., rod-shaped) bacteria is highly desirable for all investiga-tions and manipulation where dedicated control is required. This may apply for the observation of intracellular processes in which lateral observation of the bacterium is critical or for the assembly of biohybrid microsystems since a complete control of the bacterium during the adhesion process is necessary due to bacterial polarity. Moreover, independent control of multiple particles might also be needed in, for example, microfluidic applications if parallel processing of large amounts of par-ticles is required.

These demands can only and finely be addressed in an elegant way by holo-graphic optical tweezers (HOT) based on computer generated holograms [108], which enables one to tailor the light trapping beam into several single tweezers or even more complex optical traps, for example, nondiffracting and self-similar light fields using a spatial light modulator (SLM) [109]. Thus, by structuring the trapping landscape, this approach extends the possibilities of optical tweezers to the dynamic manipulation over a multitude of particles.

The unique versatility of HOT allows the rotation of elongated particles, which initially align automatically parallel to the laser beam [110,111], by using two or

426 CHAPTER 14 Controlling autonomous nanobiorobots

more traps as handles that exert the required torque [112]. Thus, rod-shaped bac-teria can be oriented along any arbitrary axis with the appropriate relative position of both traps [110]. For example, lateral orientation of rod-shaped bacteria is real-ized creating two very close optical traps and moving the one spot in the x-y plane while the other one is kept in a fixed, static position, as shown in the schematic and experimental images in Fig. 14.5. This procedure can even be applied for bacterial chains during the process of duplication bacteria by adding more traps to effectively stabilize position and orientation of the bacterium. In contrast to bacterial orientation control with dual beam optical tweezers [98], control and addition of two or more traps are achieved by simply updating the computer-generated hologram, avoiding changes in the mechanical or optical experimental setup.

Furthermore, advanced control by means of HOT is not limited to a single bac-terium, but it is also applicable for the simultaneous and individual control of large numbers of bacteria. This control allows the creation of reconfigurable bacterial mi-croarrays [113], as well as fixed arrays in gelatin matrix and in a photopolymerizable hydrogel [114–116]. Besides their prospected potential in synthetic biology, these bacterial microarrays are also exploited for the parallel analysis of the biophysical properties of multiple rod-shaped bacteria, for example, for their rotational behavior [117] or for their response to laser trapping photodamage [118]. In addition, HOT-assisted arrangement of bacteria and phototoxic microparticles have been recently

FIGURE 14.5

(A–D) Schematic and (E–H) experimental demonstration of full three-dimensional control over a rod-shaped bacterial cell (B. subtilis). (A, E) Initially, the bacterium aligns with the beam propagation direction of a single trap (black circle). (B, F) The creation of a second trap (indicated with a dashed circle) allows rotating the cell with an arbitrary angle in the x-z plane. (C, G) The bacterium is rotated until being oriented perpendicular to the z-axis. (D, H) The bacterium is rotated in the observation plane.

Adapted from Hörner et al. Full 3D translational and rotational optical control of multiple rod-shaped

bacteria. J Biophotonics 2010; 3: 468–475 [110].

4275 Optical manipulation of bacteria

proposed to define scenarios that allows to characterize the dependence of bacte-rial photodynamic inactivation on the distance between the bacterium position and a source of reactive oxygen species production [119].

Trapping light landscapes based on higher order light modes extend further the capabilities of optical tweezers, in particular for orientation control in an x-y plane [120–122]. For example, the annular profile of Laguerre–Gaussian (LG) beams can be used in biological applications for minimizing photodamage to the biological specimen since the light intensity distribution is distributed in a larger area than a fundamental Gaussian beam [123,124]. Other relevant light fields with a complex behavior during propagation, for example, Elegant Gaussian beams, enhance the ca-pabilities for trapping of abiotic elongated particles [125]. However, these optical po-tential landscapes so far have not being exploited for investigations of biomolecular motors, bacterial adhesion control and assembly of biohybrid systems. We will show in Section 6 some original and actual approaches to close this gap, paving the way for an integral biomedical light landscape HOT system.

Finally, the emergence of alternative optical trapping schemes is opening new routes for manipulation of biological specimens without the requirement of expen-sive objectives to focus the laser beam onto a diffraction-limited spot. For instance, abruptly tapered optical fibers have been used in combination with microfluidics for the arrangement and trapping of rod-shaped bacteria [126]. Similarly, surface plas-monic lenses fabricated on the end face of an optical fiber have been employed for contact-less optical trapping of bacteria [127]. In addition to these optical fiber-based approaches, silicon photonic crystal cavities and resonant optical antennas have been designed for trapping and patterning bacteria just by optical means [128,129].

5.3 PHOTODAMAGE ON BIOLOGICAL SAMPLES DURING OPTICAL TRAPPINGOptical tweezers, and optical manipulation techniques in general, are suitable for the manipulation of biological specimen, given that the applied optical forces are nondestructive and of the same order of magnitude as many relevant biological phe-nomena in the range of piconewtons. However, light absorption of the trapping beam and other collateral effects arising due to laser light irradiation have to be considered and minimized to affect as little as possible the process of investigation and to ensure cell viability.

Despite the fact that the optically induced damage or photodamage of the laser trapping beam depends strongly on the specimen under study, cell viability and survival depends mainly on the wavelength of the laser beam, the laser power at the specimen plane, the exposure time, the presence of oxygen, and on whether the cell has been prestressed [97]. Cell viability can be examined via various indicators, including cloning efficiency, fluorescent markers, and evaluation of changes or even lack of motility in the case of swimming cells, among others.

A particular important parameter to be considered in order to minimize the pho-todamage due to light absorption is the operating wavelength. For manipulation of

428 CHAPTER 14 Controlling autonomous nanobiorobots

biological specimens usually the operating wavelength of the trapping laser is cho-sen in the near infrared, where a window of transparency is found [130]. However, different tolerances have been found within the interval from 700 to 1064 nm for different cell types, including both eukaryotic and prokaryotic cells. For mam-malian cells, a maximum viability has been found for light exposure in the range between 950 and 990 nm, while maximum damage was observed between 740 and 760 nm, resulting in cell death of all examined bacteria after only 1 min of exposure to the trapping beam [97]. Similar response was found in the case of prokaryotic cells, whose maximum viability was detected at 970 nm and minima at 870 nm and 930 nm [131]. Interestingly, it has also been shown in human breast adenocar-cinoma cells that preirradiation with a 632.8 nm source increases significantly the viability of cells trapped later with optical tweezers at 1064 nm [132]. This effect can be explained by the activation of protection mechanisms of cells during the preirradiation [133].

A more intriguing parameter is the presence of oxygen during trapping. While it has been observed that anaerobic conditions or the presence of oxygen- scavenging molecules have definitely an influence on the cell viability, available studies [131,134] show contradictory conclusions regarding the loss of viability by the presence of oxygen. In any case, these studies highlight the demand to study the optical damage to specific biological specimen from case to case. The influence of other parameters, as the power of the trapping laser light and the irradiation time, have been also ad-dressed, resulting in a decrease of cell viability when these parameters are increased [118]. In conclusion, optical tweezers-induced damage in the biological sample should not be in any case totally discarded. However, a suitable selection of the pa-rameters related to this photo damage can ensure cell viability without compromising the applicability of optical manipulation in biological experiments.

6 FUNCTIONAL OPTICALLY ASSEMBLED SYSTEMS BASED ON BIOMOLECULAR MOTORSAfter having elucidated the propulsion, adhesion, and manipulation of biomolecular motors, and particularly of bacterial flagella motors, controlled assembly of biohy-brid microsystems and microrobots can be realized on the basis of HOT’s full 3D manipulation with controlled attachment on functionalized surfaces. For this purpose, suitable selection of the laser wavelength (λ = 1064 nm), power level (few milliwatts for each optical trap), and exposure time (less than 5 min) was required to ensure minimal absorption by the biological specimen, and thus preventing damage during assembly. In the following sections, an overview of actual and exciting experimental demonstrations of functional biohybrid systems that have been implemented up to now and which rely on optically-assisted assembly are presented. These included mi-crofluidic mixers (Section 6.1) as a coherent collective approach by defined surface assembly and bacterial one-to-one microrobots that carry biomedical cargo over long distances (Section 6.2).

4296 Functional optically assembled systems based on biomolecular motors

6.1 BACTERIA-BASED MICROFLUIDIC MIXERSOur first demonstration of an optically-assembled biohybrid microsystem consisted in tethering multitude bacterial cells to a surface in a controlled way for defined microfluidic flow induction [135]. For this purpose, we employed readily prepared or commercially available polystyrene coated surfaces since polystyrene does not affect the viability of adhered bacteria [82]. Following the experimental procedure for the creation of reconfigurable bacterial microarrays with HOT [113], bacteria that exhibited a significant motion in the optical traps were preselected and optically-arranged on the surface (Fig. 14.6A).

Despite only approximately 50% of the bacteria attached after a contact time of roughly 2 s, defects in the bacterial pattern could be easily filled with additional bacte-ria. More efficient binding to the surface can be easily employed by additional schemes for enhanced bacterial adhesion as those described in Section 4.3 and reviewed in detail in [66]. In this way, a wide variety of possible patterns of fully rotating bacteria has been created by tethering bacteria to the surface with exactly one flagellum.

After applying the trapping laser beam, the bacteria rotated, typically at fre-quencies of a few Hertz, around a rotation axis in close proximity to the cell body (Fig. 14.6C,E). Maximal frequency rotation at 9 Hz was observed for at least a couple

FIGURE 14.6

(A) Schematic of the optically-assisted bacterial adhesion of a self-propelling bacterium on a coated surface based on a holographic optical tweezer (HOT). (B) Bright-field image of multiple B. subtilis attached to a coated surface. The arrows superimposed on the image indicate the flow of the surrounding fluid resulting from bacterial actuation. (C–E) HOT-assisted array pattern of 2 × 4 motile B. subtilis. Each bacterium rotates with different frequencies as it can be observed by the image sequence.

Adapted from Woerdemann et al. Structured attachment of bacterial molecular motors for defined microflow

induction. Optofluidics Microfluid Nanofluidics 2014;1:19–26 [135].

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of minutes, and changes in direction of rotation occurred occasionally and abruptly from counter-clockwise to clockwise or vice versa. Moreover, the stop-resume se-quence of the rotation can be controlled by HOT-generated traps at specific positions and with a minimal power level to avoid photo damage.

In order to investigate the potential of these bacterial-rotating microarrays for induction of fluid dynamics at low Reynolds number, we employed fluorescent polystyrene beads as velocity sensors after the arrangement of the bacteria with optical tweezers was performed. Used as nondisturbing, transparent tracers, these fluorescent nanoparticles were imaged by fluorescent microscopy, and subsequently their motion was analyzed by classical particle image velocimetry. As a result, the impact of the bacteria rotation on the fluid was found to be a defined circular flow with slightly varying velocities (Fig. 14.6B). This can be attributed to the fact that individual bacteria have different motilities and that the bacterial rotation frequency of a single bacterium varies over time.

In addition, this procedure allows determining the interbacterial distances needed to achieve strong interaction between individual cells, resulting in a range of about 2 µm around the rotational center. Thus, these results demonstrated clearly that self-propelled bacterial rotational motors can be arranged on surfaces with HOT at defined positions in such a way that they can be used as strong fluid mixers, guide motors for micro- and nanoparticles, or induce dynamic fluid motion based on col-lective coherent addition of forces. More sophisticated bacterial microfluidic flow mixers with an even more efficient cooperative behavior between cells is possible by choosing specific bacterial strains with desired rotational properties [136,137].

6.2 BACTERIAL MICROROBOTSDue to HOT’s precision and versatility to dynamically control and arrange tens of nonspherical particles in any desired configuration, HOT-assisted bacterial attach-ment to surfaces was further applied for the fabrication of biohybrid microrobots [138]. In comparison with established self-assembling schemes, HOT-assisted as-sembly enables to set the desired number of attached bacteria to the propelled mi-croscopic particle. In addition, since HOT provide full 3D position and orientation control with nanometer precision, each single bacterium can be attached with a high degree of accuracy on the surface of the payload particle. In this way, any desired configuration for assembling the microrobot can be achieved so that site-specific modification or patterning of the abiotic particle surface is no longer required for a precise attachment.

With HOT, tailored optical trap configurations can be created for the parallel manipulation in three dimensions of elongated particles and rod-shaped bacteria [111,113]. Thus, in a first proof-of-principle study, swimming B. subtilis and elon-gated zeolite L crystal were used as model of the biological and abiotic compo-nents, respectively. Based on the results of former studies, zeolite L crystals were functionalized with a monolayer of amino groups to promote noncovalent binding of bacteria on their surfaces. In addition, zeolite L crystals with 2 µm in length and

4316 Functional optically assembled systems based on biomolecular motors

1 µm in diameter were synthetized because elongated particles with a strong asym-metry are easily manipulated with HOT [139].

For the optically-assisted assembly, multiple dynamic optical traps were generated for trapping, aligning (Fig. 14.7A,D), and approaching a single zeo-lite L crystal to an optically motile bacterium (Fig. 14.7B,E). When they were in close contact, the trapping laser was turned off and both parts remained together forming the biohybrid microrobot. After HOT-assisted attachment of the zeolite L crystal to the bacterial body was realized, the bacterium propels its payload par-ticle as a universal microshuttle (Fig. 14.7C,F). The subsequent propulsion of the living microrobot, with typical velocities around 10 µm/s, indicates that no seri-ous damage is induced to the cell during the short time required for the assembly process. Noteworthy, the trajectories of the bacteria-zeolite L microrobots do not feature the characteristic straight trajectories of self-propelling bacteria during its “ running” mode. Instead, they show a curved one which might be explained by the asymmetric configuration of the bacterium-zeolite machine. In addition, a relative strong adhesion keep both particles together since separation of the microrobot blocks is not possible neither by using optical traps nor by dragging the zeolite L in the sample.

As a vision, the exploitation of more versatile surface modifications can lead to the formation of more complex biohybrid microrobots. Recently, we have developed a novel type of zeolite L based polymer brush, which features a soft surface with a high density and variety of functionalities [81]. These zeolite L/polymer hybrids support a more efficient attachment of the crystals to other particles and to bacteria

FIGURE 14.7

(A–B) Schematic and (D–E) corresponding microscopy images of the assembly of a motile bacterium (B. subtilis) and a zeolite L nanocontainer. (C, F) Subsequent self-propulsion of the biohybrid microrobot through the medium when the trapping laser is off.

Adapted from Barroso et al. Optical assembly of bio-hybrid micro-robots. Biomed Microdevices

2015;17:1–8 [138].

432 CHAPTER 14 Controlling autonomous nanobiorobots

in comparison to the monolayer systems studied, and thus they will revolutionize the assembly of more complex biohybrid microrobots for a multitude of biomedical health and high-throughput noninvasive medical analyses approaches.

7 CONCLUSIONSThe employment of biological nanomachines to perform tasks that we design to our benefit has left the stage of dream vision and has started becoming a realistic, highly promising strategy for future applications. The progressively improved control of biomolecular motors in microscopic environments has already permitted not only impressive first proof-of-principle demonstrations, but also remarkable applications in microfluidics and transport of particles at low Reynold numbers. In a step be-yond, the development and optimization of novel methods is the actual remaining challenge in order to achieve a defined control of the adhesion of the autonomous biomotors to the artificial microsystem. This will open future avenues for the fabrica-tion of autonomous biohybrid microsystems and microrobots and will considerably expand the field of micro- and nanorobotics.

Based on their flexible control of micro- and nanomater, optical tweezers-based approaches are the method of choice for prestructuring of bacterial nanomotors by precisely positioning them on the abiotic system. Thus, these methods pave the way to a customized design of complex biohybrid micromachines, for example, microro-bots with a specific number of optically arranged bacteria and abiotic particles. As a visionary step further, optical-assisted hybrid assembly can be applied to functional particles and bacteria driven by various directional control mechanisms in order to achieve a tailored higher level of sophistication. For example, multitaxes directional control could be employed, either simultaneously or switching between multiple taxes (e.g., chemotaxis-magnetotaxis, magnetotaxis-phototaxis) depending on the demand, for propelling the biohybrid microsystem.

Finally, further demonstrating the universality of HOT-assisted assembly, this approach will introduce a paradigm change in all biological relevant microenviron-ments and nanostructures, for example, it has been recently shown for fabricating three-dimensional structures of embryonic stem cells [140] and microtubule net-works [141].

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441

AAcousto-optic deflector (AOD), 35, 132Adenosine triphosphate (ATP), 413Advanced optical control, 425–427Aerotaxis, 416Amino-polystyrene beads, FITC assembly on, 222Amorphous silicon, 303Analog to digital converter (ADC), 358Angular momentum, 141, 173

density, 100electromagnetic, 102flux, 100intrinsic, 100optical, 109spin, 141vector component of, 100

Anisotropic trapping stiffness profile, 90Antireflection coatings, 112AOD. See Acousto-optic deflector (AOD) Arbitrary elliptical polarization, 104Archimedes-screw-shaped mixer, 43Assembling techniques, 43–52

advantages, 44chemical bonding, 45–46disadvantage, 44interlocking connection, 49–52joining by polymerization, 48–49microstructures applications, 37, 53–58

magnetic microrotor, 54–56directed fluid flow, 56field determination and pumping, role in

assembling, 54–55flow field determination and pumping, role

in, 54–56flow field measurement, 56

microrotor assembly using screw connection, 57–58

thermal and photothermal connection, 46–47Atomic force microscopy (AFM), 88, 271, 347Automated cell shape detection, 394–397, 405Automatons, 265Autonomous microrobot, 268Azimuthal mode index, 122

BBacillus subtilis, 414Bacteria-based microfluidic mixers, 429–430Bacterial adhesion, 419

control of, 422–423

role of cell surface structure mediating, 420–422theory of, 419–420

Bacterial carpet, 417Bacterial microrobots, 418–419, 430–431Bacterial microsystems, 417Bacterial patterning, 419

control of, 422–423Bacterial-powered microfluidics pumps, 417–418Bacteriophage lambda DNA, 369Basic optical tweezers system, 6Bessel beams, 111, 358Bilateral control system, block diagram of, 200Bilateral teleoperation, 204, 230Biochemical assembling technique, 56Biochemical stimuli, 398Biocompatible sensors, 220Biohybrid microrobots, 36, 172Bio-(inspired) microscopic motors, 413–414Bioinspired robotics, 314Biological specimen, 423Biomedicine, 411Biomolecular motors, functional optically

assembled systems, 428Biomotors, 412Biophotonics workstation, 49Biorobotics, 314Biotin, 80Bipolar perturbation, 401Bipolar stimulus, 400Birefringent material, 101, 105, 168Birefringent optical properties, 349Birefringent particle, 142Boltzmann constant, 8, 214Brownian motion, 8, 121, 157, 214Bubble oscillation, 303

CCaenorhabditis elegans, 386Canonical stress, 102Carbon-based photothermal functionality, 305Carbon nanotubes (CNT), 194, 206, 208, 238Caulobacter crescentus, 422Cavitation-bubble approach, 294Cell-cell fusion, 314

induced by optically heated GNPs, 335Cell-GUV fused structure, 333Cell manipulation, 194, 206Cell mask plasma membrane stain, 229Cell migration, 385

Index

442 Index

Cell puncture, 231Cellular dynamics, 266Cellular sensing, 386Cell viability, 427, 428Centroid values, for trapping points of probe, 364Charge-coupled devices (CCDs), 358Chemical functionalization, 34Chemically loaded PLGA microsources, 390Chemical microsources

fabrication and characterization, 389Chemical vapor deposition techniques, 305Chemotactic cell response, 397

fMLP gradient, 398HL-60 neutrophils, 397induced chemotaxis, by optically manipulated

chemical microsource, 399local chemical gradients, 397–398local chemical perturbations, 398–400

bipolar perturbation of cytoskeleton, 401splitting of a lamellipodium in response

to a, 402quantitative data analysis, 398

Chemotaxis, 386, 389, 416induced, by optically manipulated chemical

microsource, 399Chemotaxis, 385Chip-based systems, 24CMOS sensor, 362CNT. See Carbon nanotubes (CNT) Complementary metal-oxide semiconductor

(CMOS) camera, 358, 359Complex polymer microtools

applications, 172–184functionalized structures, 182–184nonfunctionalized structures, 172–182

Composite hologram, 199Composite particles

with two conical sections, joined by cylinder, 75Computer-generated hologram (CGH), 425

generator, 200Constant force surface image recording

with microtool, 92Contact-free optical techniques, 287Coulomb interactions, 150Cycle-averaged optical force, 73Cylinder, negligible horizontal axial trap

stiffness, 75Cytochalasin D (CytoD), 390, 397, 402

concentrations, 400lamellipodium in response, 402loaded microsources, 398

Cytoskeleton, 401biomolecular motors, 425

DDDA. See Discrete dipole approximation (DDA) Delivery/capture efficiency, 297–298Dendritic cells, 314DFS. See Dynamic force spectroscopy (DFS) 4′,6-Diamidino-2-phenylindole (DAPI) dye, 252

images of fluorescence signal in, 253Diatom algae

optical image, 85SEM image, 85silica shelled, 85

Dichloromethane (DCM), 390Dictyostelium discoideum, 386Dielectric permittivity, 73Differential interference contrast (DIC)

microscopy, 394application, 394Gaussian filter, 395imaging, 394, 405

HL-60 cell, 396J774A.1 macrophages, 397

MATLAB-function regionprops, 397segmentation of a single cell, 396

Diffractive optical element (DOE), 171Digital micromirror devices (DMD), 155Digital microscope camera, 11Dipoles, 319Direct cell targeting, 294

diode laser sources, 295microfluidic approaches, 294single-cell precision, 295third-harmonic beam, 295trypsinization, 294

Directional control methods, 415Direct laser writing (DLW), 82Direct optically triggered membrane

effects, 289detergent solubilization, 289interactions, coherent light and biological

materials, 289mechanisms of action, 290–292

formation of range, of lipid membrane bodies, 291

low-density plasma formation, 290power density, 290single cell-poration and transfection

technique, 292thermal fluctuation, 290

Discrete dipole approximation (DDA), 116Disruption of cell membranes, 288DLVO theory, 420DLW. See Direct laser writing (DLW) DMD. See Digital micromirror devices (DMD)

443Index

DMEM. See Dulbecco’s Modified Eagle’s Medium (DMEM)

DNA-DNA linkage, 45DNA double-stranded break repair

homologous recombination (HR)-mediated, 25DNA repair protein UvrA, 370DNA scanning probe, 348DNA segment for testing, 348DNA tethered, 350DNA tightropes, 369, 373, 377

detection of forces from, 371probe scanned along, 372

and bound protein, 373for single protein molecule, illustration of

experiment, 373DNA uncoiling, 347DOE. See Diffractive optical element (DOE) Dulbecco’s Modified Eagle’s Medium (DMEM), 157Dumb bell, 348Durotaxis, 385Dye excitation, 252–255

DAPI dye, 253guided SH and excited fluorescence signal vs

incident laser beam power, 255imaging resolution, 254procedure of sample preparation, 253signal-to-noise ratio (SNR), 254single-photon absorption (SPA), 254smallest cross-section and crystal structures of

nanowires, 256SNR of fluorescence signal vs average power of

guided SH, 255threshold power of near-infrared laser light, 254two-photon absorption (TPA), 254

Dynamic force spectroscopy (DFS), 132

EElectric fields, 416Electric tweezers, 129, 150, 151

plasmonic-active nanomotors operated by, 154rotary nanomachines operated by, 152

Electroformation, 326Electromagnetic coils, 55Electromagnetic energy functional, 69Electromagnetic waves, 129Electron beam lithography (EBL), 168, 351, 352Electron multiplier charge coupled device

(EMCCD), 361Electro-optic deflector (EOD), 35, 132Electrophoretic forces, 150, 416Electroporation, 317Electrorepulsive interaction, 423

Elodea densa, 137Endocytosis, 314Energy barriers for fusion, 316Energy density, 316Enhancement factor (EF), 153Entropy, 395Environmental conditions, 415EOD. See Electro-optic deflector (EOD) Equipartition theorem, 346Escape force measurement, 365Escherichia coli, 414Ethylenediaminetetraacetic acid (EDTA), 416Eukaryotic cells, 427Exopolymeric substances (EPS), 421External noise sources, 7External optical train, 11

FFabry-Perot modes, 238F1-ATPase based nanopropellers, 414, 416Faxen’s law, 365FDTD. See Finite difference time domain

method (FDTD) FEM. See Finite element method (FEM) Femtosecond laser, 39, 116, 206–208, 215, 217,

218, 296ablation, 215, 217

Feynman, Richard, 129FH. See Fundamental harmonic (FH) FIB milling. See Focused ion beam (FIB) milling Filament-like track structures, 350Filopodia, 314, 394Finite difference time domain method (FDTD), 116Finite element method (FEM), 116First-order beam, 199FITC. See Fluorescein isothiocyanate (FITC) Flagellar molecular motors, 412Flagellated bacteria, 415Flat optical restoring force

microtool engineered, 92Flexible microtools, scanning electron

micrographs, 94Flow cell, 354Flowmeter, 119, 120Fluorescein isothiocyanate (FITC), 221, 223

relative fluorescence intensity of, 224Fluorescence microscopy, 348Fluorescent chemical labels, 348Fluorescent dyes, 221, 229

YOYO-1, 369Fluorescent markers, 427Fluorescent nanobeads, 229

444 Index

Fluorophores, 182, 323Flux densities, 100Focused Gaussian laser beam

particle at different places, 5Focused ion beam (FIB) milling, 241Force measurement

using image analysis of microscopic camera images, 201

Force probe calibration, 362calibration of force probe position, 363

AOD calibration value, 363region of interest (ROI), 363

initial calibration of laser position, 362trap stiffness measurement, 363–365

Force probe design, 350–351probe production processes, 351–352probe release and loading into solution, 353–354

Force probe image, 360experiencing motion, diagram, 376using two-photon polymerization, 353

Force transducer, optical image, 82Form birefringence, 142Formyl-methionine-leucine-phenylalanine (fMLP),

390, 397Fourier hologram, 198, 199Fourier plane, 199Fourier transform, 9Fresnel equations, 130Fresnel formulae, 170Fresnel lens, 199Fundamental harmonic (FH), 242

GGalvanotaxis, 416Gaussian beam, 104, 107

laser, 11profiles, 52

Generalized Lorenz-Mie theory (GLMT), 116Generalized phase contrast (GPC), 87, 195, 268, 358Generated 3D microcomponents

CAD file, 52Gerchberg-Saxton method, 200Giant unilamellar vesicles (GUVs), 317Gliding assay, 415GLMT. See Generalized Lorenz-Mie theory (GLMT) Gold, 299

chemical switching of nanoparticle aggregation, 300

inertness, 299manipulation of cell membranes, 299nonstick coating, 299optical heating, 301

plasmonic heating, 300production of multifunctional nanomaterials, 299toxicity, 302

Gold nanoparticles, 182, 319Gold seeding, 45Golgi network, 314GPU. See Graphics processing unit (GPU) Graphics processing unit (GPU), 195Gratings, 88Guided SHG signal, 247

adjustment of nanowire length, 249–252conversion efficiency of 1st FH mode, 251intensity profiles of modes, 251phase-matching effect, 249SH power generated, by 1st FH vs nanowire

length, 250vs nanowire length, 252

modal phase-matching in LiNbO3 nanowires, 247result of measurement, 248SEM image of studied nanowire, 247vs. incident laser wavelength, 248wavelength-dependent Sellmeier equations

for, 248

HHamamatsu Photonics X10468-03, 197Haptics, 22–24

coupling loop, 23tools, 22

Head tracking algorithm, pseudo 3D display, 22HeLa cells, 157Helmholtz equation, 103Hemifusion, 316High-performance liquid chromatography

techniques (HPLC), 390Holey spheres, 178Hollow microrobots, for material transport, 273

applications in drug delivery, 273counter-propagating beams, 275deposition process, 274ejection of cargo, 277fabrication protocol, for metal-embedded

microrobot, 274flow speed of thermal current, 276layer-by-layer fabrication, 273parametric equations, 273position of heating beam, 277scattered silica beads, 275surface tension gradient and Marangoni

convection, 275two-photon fabrication setup, 273two-photon polymerization, 273

445Index

Holographic microscopy, 20Holographic optical trapping (HOT), 15,

387, 430application for live cell experiments, 389freely movable microstructures, 51

Holographic optical tweezers system, 35, 425. See also Light robotics

advanced control by, 426assisted arrangement of bacteria and phototoxic

microparticles, 426integral biomedical light landscape system, 427precision and versatility, 430with stereoscopic tracking arm, 87typical configuration, 44typical set-up, around inverted microscope, 87

Homoeostasis, 314Hookean spring, 7Hooke’s law, 346Human interfaces, 15–16

optical manipulation systems, software control of, 15–16

Hybrid nanorobots, 195, 211, 229, 305cell puncture, 231design of, 213fabricated, 216fabrication process of, 209with functional nanomaterial, 207optical manipulation of, 218

Hybridoma technology, 314Hybrid T-matrix/DDA method, 111Hydrodynamic interactions, 180Hydrodynamic resistance, 77Hydrodynamic synchronization, 178Hydrogen bonds, 422

IImage processing, 16, 201, 202, 395

post processing of, 394imfill function, 395Inductive coupled plasma reactive ion etching

(ICP-RIE), 241Integrated imaging techniques, 37Integrated optofluidics, 35Intercellular communication, 314Internet

accessible platform interactive collaboration, structure, 25

controlling systems remotely, 24–25Ion-beam enhanced etching (IBEE), 241Ionic polymerization, 40Isometric clamps, 133Isotonic clamps, 132

JJ774A.1 cell line, 387Jurkat cells, 157

KKinoform hologram, 197KNbO3 nanowires, 240

LLab-on-a-chip systems, 176Laboratory-on-a-chip (LOC), 34LabVIEW, 16Laguerre-Gaussian (LG) beams, 100, 109, 119,

144, 427VSWF and, relationship between, 103

Lamellipodia, 394complete detection of, 395Gaussian filter applied to thin boundary, 395retraction of, 400, 401shape and dynamics, 400splitting, 398, 402thin protrusions containing, 400

Langevin equation, 8Laser-assisted polymerization, 175Laser beam, 427Laser hologram generation, 217Laser trapping power, 15Laser tweezers, 169, 194Lenses, 88Light-driven microrobots, 268

harnessing, from optical trapping and micromanipulation, 268–270

additive manufacturing, 270cellular deformability, 268fabricated microbeads, 268optical forces, 268optical manipulation, of plurality of

microbeads, 269optical momentum transfer, 270spring-mass microsystem, 270subtractive manufacturing, 270two-photon fabrication systems, 270

turning microstructures into functional microrobots, 270–272

basic elements, 270basic illustration, of functional load, 271functional load and trapping handles, 270

Light-driven systems position and force measurement, 6–9

drag force method, 7equipartition, 8

446 Index

Langevin method, 8–9light deflection method, 9

Light robotics, 266biomedical applications of, 229–233

contact force of microrobot to red blood cells, 229

overview, 229single cell puncture by using laser heating of

CNT, 229–230virus-infected cell membrane, measurement

of pH, 231–233fabrication technology for, 206–218

3D hybrid nanorobot integrating functional nanomaterials, 206

hybrid nanorobot fabrication of, 208–215manipulation of, 215–218

overview, 206system configuration, 207

multiple-trap manipulation system for, 195–205overview, 195–196system configuration, 196–202teleoperation system, evaluation of, 202–205

scaling of physical effects, 266characteristic length, 266measurement, of electrostatic force, 267scaling, of attractive forces, 267surface related properties and effects, 266volume-related properties and effects, 266

sensing technology for, 220–228materials and method, 222–223optical multisensing microrobot, 221

concept of, 221evaluation results of, 223–226

overview, 220–221tools

schematic representation of, 288for single cell biochemistry and

biophysics, 289tunable surface properties, 289

LiNbO3 nanostructures, 239LiNbO3 nanowires, 238

for biological applications, 254fabrication of, 240

bottom-up chemical synthesis methods, 240–241hydrothermal method, 240sol-gel method, 240top-down fabrication methods, 241, 242

Lipid bilayers, steps in fusion, 317Lipid membranes, 350Lipopolysaccharides, 421

Liquid crystal display, 197Lithium niobate nanowires, 2083D-Lithography processes, 38Living cells fusion, 334

controlling, 334optimizing, 337verifying by lipid and lumen mixing, 334–335viability after, 336

Localized surface plasmon (LSP), 145Lorentzian curve, 9Lorentzian fit, 9Lorentz invariance, 102Lorenz-Mie theory, 112LSP. See Localized surface plasmon (LSP)

MMachine, 99

definition, 99Madin-Darby canine kidney (MDCK) cells, 229, 232Magnetic anisotropy, 153Magnetic microrotors manufacturing

chemical bonding, 55Magnetic trapping (MT), 347Magnetotactic bacteria, 418Magnetotaxis, 416Manipulation force, 212Manufacturing complex microstructures, 43Mass manipulation, 195Maxwell stress tensor, 68

method, 6Mechanical manipulation, 167Mechanical microgrippers, 36Medical robotics, 266Membrane fusion, 314

events occurring inside cells, 315Metallic nanoparticles, 319

optical confinement of, 319–322Metal powders, 37Microbeads, 168, 184, 196, 214

based devices, 134polystyrene, 222

Microchannels fluid flow control, role in, 53implemented microrotor, schematic depiction, 56

Microcomponents, cross-section, 52Microdisc resonators, 169Microelectrodes, 155Microfabrication, 3D

biochemical bonding, 46Microfluidics, 33–37, 168, 178

chips, 33, 215

Light-driven systems (cont.)

447Index

contents, 37definition, 34devices, 99, 118, 175

manufacturing, 36light-based, 35manufacturing, 34materials, 34metallic structures in, 272–273

Förster resonance energy transfer (FRET) microscopy, 272

laser-induced heating, of thin metallic layers, 272

surface-enhanced Raman spectroscopy, 273thermal effects, 272

microstructures assembling of, 36–37techniques, 294

Micromachines, 101Micromanipulation, 194

techniques, 424Micromanipulator, 177Micro mechanical systems, 37Micromotor, 176Microparticle targeting, 305

effects of topography, on nanoparticles, 306Microreactors, 34Microrings, 50Microrobots, 221, 227

3D microrobotic systems, 49with functional loads, 272

chemical sensing, 277–278hollow microrobots, for material transport,

273–277metallic structures, in microfluidics, 272–273for temperature sensing, 279–280thermal application of, 279

Microrotor, 139, 140assembling process, 57

SEM image, 58using screw connection, 57–58

magnetic, 54–56assembling magnetic rotors with different

shapes, 54–55directed fluid flow, 56flow field determination and pumping, role in,

54–56flow field measurement, 56

Microscopy, 4, 5, 7, 10, 12, 15, 19, 20, 84, 86, 90, 93, 106, 121, 123, 169, 175, 181, 184, 195, 207, 217, 238, 239, 245, 271, 273, 287, 319, 328, 331, 346–348, 350, 354, 358, 361, 362, 365, 379, 387, 392, 424

Microsensors, 221Microsoft Kinect, 20, 21Microspheres, translational motion, 66Microstructures

generation with two-photon polymerization, 37–43

perforated compartments, 3D SEM picture, 51Microtool, 66

construction, by directed assembly, 82fabrication, 80–88

direct laser writing (DLW), 82–843D optical control, 86–882D photolithography, 813D tracking, 85–86naturally occurring microtools, 85in situ directed assembly of components,

80–81using direct laser writing

SEM images, 84Micro total analysis systems (µTAS), 34Microtransport, 415Microtweezers, 176Microvalves manufacturing

polymerized joining, 53Mie scattering pattern, 20Mie scattering regime, 130Mie scattering theory, 6Mie theory, 324Miniaturization, 413, 418Miniaturized machines, 129Mobile light guide, 181Molecular fusion machinery, 315Monoclonal antibodies, 314Multibeam generation methods, 195

computer-generated hologram (CGH) method, 195

generalized phase contrast (GPC) method, 195

time-shared scanning (TSS) method, 195Multiforce-sensing, 205Multitouch console, 19Multitouch devices, 18Multitrapping, 205Muscle contraction, 345

NNaNbO3 nanowires, 240Nanoengineering, 411Nanomaterials, 206, 238Nanometric video tracking, 81Nanomotors, 412

448 Index

NanoNewton, 167Nanooptical systems, 16Nanorobotic systems

3D control, 19–21demonstrations or collaborative working, 18ease of use, 17enhanced user experience, 17extra information

given by user, 18received by user, 18

manipulation, 207optical manipulation of, 218peripheral devices, control with, 16–19specialist tools, 18

Nanorod, 42motors, 149

Nanoscale plasmonic motors, 150Nanoscribe’s system, 169Nanowires, 70, 71, 77, 135, 149, 151, 153,

238–241, 244, 245, 249, 250, 252, 254, 256, 279, 280, 306

optical application of, 239–240coupling and waveguiding of light, 239development, of optical logical

elements, 239lab-on-chip applications, 239second-harmonic (SH) light, 239

Near-field nanoplasmonic traps, 69Near-infrared poration, 295–297

diffraction-limited spot, 295diffraction-limited trapping beam of, 297earth’s equatorial surface, 295laser-scanning microscopy, 296photochemical effects, 297quantum mechanical effect, 296two-photon absorption, 297

efficiency, 296phenomena, 296

Negative photoresist, 208NEMS devices, 153Neutral density filter (ND), 197Newton forces, 345NIR lasers, 318Nitzshia subacicularis, 85Noether’s theorem, 102Nonabsorbing/isotropic/dielectric particles

optical force and torque densities, 74Nonbirefringent materials, 142Nonspherical particles, equilibrium trapping

orientations, 72Novel imaging techniques, 88Nuclear fuel rods, 22

OOAM. See Orbital angular momentum (OAM) On-chip cell analysis, 221On-chip robots, 229Optical angular momentum, 100–102Optical assembling techniques, 36Optical control, 314

plasmonic nanoheaters, 319experimental details, 319metallic nanoparticles, optical confinement

of, 319quantification of nanoscale heating on

membranes, 322–325temperature near an irradiated nanoparticle, 322

selective cell-cell fusion, 314Optical force, 45, 346Optical fountain, 4Optical gradient force, 4Optically controlled fusion, 314, 317–319Optically controlled microtools, 80Optically driven micromachines, 107, 109Optically driven micro/nanorobots, 194

cell manipulations with, 195overview, 194–195

Optically trapped microspheres, 88Optically trapped particle, 7Optically trapped rotator, 93Optical manipulation systems, 10–15, 170–172,

314. See also Light roboticsactuators

actuating structures, 170–172fast steering mirrors, 11galvanometer scanning mirrors, 11microscope stages, 11open-loop control, 14

advantages, 14piezo-mirrors, 11quality imaging, 11rotary stages, 11sensor-based piezo actuators, 11system design, 10–12system implementation, 12–15

of bacteria, 423feedback control mode, 13hardware system, block diagram, 13key components, 14main components, 10

actuating devices, 11actuators, 11camera, 11computer, 11microscope, 10

449Index

sensors, 12high-speed cameras, 12quadrant photodiode detectors (QPDs), 12

micromanipulation, 168Optical momentum, 130, 138Optical restoring force, 91Optical scanning probe microscope, 67Optical screw-wrench, 52Optical torque, 137Optical transport, 43Optical trap exist, 5

fiber, 5Optical trapping systems, 10, 50, 133, 134, 170,

176, 347, 385, 424holographic, 171, 172manipulation of nanowires by, 135microscopic particles, 424–425microstructures driven by, 136photodamage on biological samples during, 427

Optical tweezers (OT), 35, 100, 110, 119, 130, 139, 144, 176, 178, 194, 206, 215, 218, 232, 386, 405, 427. See also Light robotics

advantages, 35for micromanipulation, 194

basics, 4–6forces, 6optical gradient force, 4optical tweezers, 4practical setup, 5

computer-controlled system, 24toolbox, 68

holographic, 185, 195, 198femtosecond laser exposure and, 207optical system of, 198teleoperation system using, 197

induced damage, 428manipulation mechanisms of, 131multiple point manipulation with, 196particles, force density, 74plasmonic, 147

micro/nano rotary motors driven by, 148for single-biomolecule investigation, 132user interface of, 196

Optimal performance factors fast rise time, 14less overshoot, 14short settling time, 14

Optoelectronic tools, 304Optoelectronic tweezers, 129, 155, 156

development of, 158virtual micromachines enabled by, 157

Optofluidics, 33

Optomechanical microtools applications, 88–91design of, 66

Orbital angular momentum (OAM), 141trapping and rotation of microparticles by, 145turbine-like microrotor, rotation of, 146

Orthogonally orientated microscope, 20

PPaddle wheel, 110

driven by linear optical momentum, 141titania, 112

Particles with different rotational symmetries cross-sections for, 71

PBS. See Phosphate-Buffered Saline (PBS) Peak forces measured vs. angle of DNA

tightrope, 378Pediastrum

surface images, by optically trapped diatom, 89Phagocytosis, 387Phase-mismatching, 243Phase-only spatial light modulator, 87Phase transition temperature, 318Phosphate-Buffered Saline (PBS), 157Photochemical reactions, 48Photodamage, 427Photolithography, 2D image, 80Photon flux, 41Photonic crystals, 48Photonic force microscopes, 22, 346Photon momentum, 130Photopolymerization process, 37, 133

chain propagation, 38photoinitiation, 38termination, 38

Photoresist, 81Photosensitive materials, 37Phototaxis, 416PicoNewtons, 130, 133Pinned cross-rotors, design/production of, 118Plasma-assisted chemical vapor deposition

techniques, 303Plasma membrane, 314Plasma shockwave generation, 292

capture and downstream analysis, 294cavitation-bubble approach, 294limitations, 293microfluidic isolation, 294positioning of pulsed beam, 293ultrasonic experiments, 293water cavitation, 292

450 Index

Plasmonics, 129Polarization, 141, 320

laser beam, 197Polydimethylsiloxane (PDMS), 34, 302, 419Polyethylenimine (PEI) reagents, 297Poly(lactic-co-glycolic acid) (PLGA), 389

fabrication of microsources, 390microspheres, 389

Polymer powders, 37Poly(methyl methacrylate)(PMMA) microparticles, 47Polyplexes, 297Polystyrene particles, 114, 397Polyvinyl alcohol (PVA), 119, 390Pomatoceros lamarckii, 296Porous media, 415Possible microtool geometries

selection of, 79Powering microsystems, 412Poynting vector, 1022PP. See Two-photon photopolymerization (2PP) Primary initiator particle density, 42Probe displacement, 367Probe production processes, 351Probe release, 353–354Probe scan, parallel/perpendicular forces, 377Prokaryotic cells, 427Protein-DNA interactions, 345Pure membrane systems, fusion, 326, 330

cell viability after cell-vesicle fusion, 332–333controlled cell-vesicle fusion, 331controlled vesicle-vesicle fusion, 327verifying fusion by mixing of lipids and vesicle

lumens, 328verifying lipid and lumen mixing in, 331–332

PVA. See Polyvinyl alcohol (PVA)

QQuadrant photodiodes, 23, 86Quantum dots, 373

RRadical photoinitiator, 48Raman excitation laser beams, 182RATTS. See Real-time automated tracking and

trapping system (RATTS) Rayleigh approximation, 68Rayleigh criterion, 10Rayleigh equations, 67Rayleigh regime, 7Rayleigh scattering, 130RBC. See Red blood cells (RBC) Real-time automated tracking and trapping system

(RATTS), 14

software control, 17Recording surface topography

using optically trapped microtool, 91Red blood cells (RBC), 229

stiffness measurement of, 229Refraction forces, 103Refractive indices, 114Resultant microtools, 83Reversible bacterial adhesion, 423Reynolds number, 7, 34, 77, 101, 363, 413, 414, 430Rhodamine-B (Rh-B), 46, 221, 223, 224RMSE. See Root mean square error (RMSE) RNA polymerase, 351RoboLase IV screen, front panel display, 26Robotic micro/nanodevices

electrically and optoelectronically enabled, 150–157

electric tweezers enabled, 150–153optoelectronic tweezers enabled, 155–157

optically enabled, 130–144nanomanipulation and robotization with

controlled photon momentum, 137–144optical traps, 131–137

plasmonically enabled, 145–149Root mean square error (RMSE), 121Rotary stepper motor, 15Rotary torque, 144Rotating machines, 99Rotating micromachines, optically driven

applications, 117–122pinned cross-rotors, case-study, 118–122

computational modeling, 116fabrication, 116–117overview, 99–100principles of design, 102–116

controlling reflection, 110–116discrete rotational symmetry with P = 2,

104–106discrete rotational symmetry with P > 2,

106–108importance of symmetry, 102–104no rotational symmetry (P = 1), 109using reflection to generate torque, 109–110

Rotational motion, 99Rotation, external magnetic field, 55Rotation speed, 151Rotor rotation, 122, 123Rotor with twisted arms, 120

SSacrificial layer, 119Sample fabrication, 244–245Scanning electron microscopy (SEM), 390

451Index

Scanning probe microscope (SPM), 88Secondary target techniques, 298–299

gold films, 299–301organelle specific targeting, 301toxicity, 302

Second harmonic (SH), 242Second-harmonic generation (SHG), 238

applications of, 238demonstration of, 246

beamspot, 246central wavelength, 246coupling efficiency of laser light, 247laser beam, 246

energy diagram of, 242experimental setup, 245

electron-multiplying charge-coupled device (EMCCD) camera, 245

schematic of, 245schematic of laser coupling, 245

and phase-matching, 242–244behavior of SH, along propagation

distance, 244efficiency of, 244wave-vector mismatch, 243

polarization density of, 242second-order nonlinear optical effect, 242waveguiding and generation of, 238

Self-assembling process, 45Self-propelled microscopic robots, 172Semiconductor nanowires, 238Sensing technology, 3D, 21SERS. See Surface enhanced Raman

scattering (SERS) Servo microscope stage controllers, 13Shape-induced optical forces, 67–76

Mie regime force and torque calculation, 68–69

discrete dipole approximation (DDA), 68finite-difference time-domain (FDTD)

approach, 68T-matrix method, 68

nonequilibrium optical forces, 72–74nonspherical particles, equilibrium trapping

of, 69–72optical tweezers, nonconservative forces in, 75–76Rayleigh regime, 67–68

Silanization reagent, 302Silica bead, in water

optical trap, power spectral density plot, 9Silica microrod, 81Silica nanoparticles, 183Single-beam gradient trap, 4

construction, 5Single cells, local stimulation of, 385–386

Single molecule experiments, 355additional components, 361application of force probes in, 345comparison of forces in, 347energies involved in, 346force experiment, 369–377methods for creating multiple traps, 357–358

two-dimensional beam steering, 357optics and laser for trapping, 355–357

forces acting on a particle, 356position measurement, 358–361

centroid calculation, 361method for accessing rows of pixels, 359regions of interest for trapping point of probe, 360

schematic of experimental system, 355Single molecule level, 346

measurements at, 346–347Single molecule optical trapping experiments,

348, 349Single-photon absorption (SPA), 254Single-photon polymerization, 38SLM. See Spatial light modulators (SLM) SNARE protein, 316, 317Solid-liquid interface, 417Solid-phase approach, 298Spatial filter (SPF), 199Spatial light modulators (SLM), 35, 171, 197,

214, 425phase-only, 87

Spatial resolution, 215Spatiotemporal resolution, 411Speed regulation, 416–417Sperm swimming force, 16SPF. See Spatial filter (SPF) Spherical nucleic acids, 299

nanometer-dimension core structure, 300Spherulites, 105Spin angular momentum, 100, 104Spin flux, 101Spiral chirality, 139SPP. See Surface plasmon polariton (SPP) Stable manipulation condition, 213, 214Stable screw connection, by OT assembling

SEM picture, 52Standard Köhler illumination, 5Stepping assay, 415Stereolithography, 38Stereomicroscopy, 86Stiffness matrix, 79Stimulated emission-depletion lithography, 42Stimulus-response mechanisms, 416Stokes drag, 203

force, 7formula, 105

452 Index

Streptavidin (SA), 45, 80SU-8 polymer, 142, 209, 210, 218, 352Surface enhanced Raman scattering (SERS), 153,

182, 277–278photoreduction of silver nitrate solution, 278practical limitation of, 277probes, 278Raman tweezers, 277structure, 184

Surface plasmon polariton (SPP), 145Surgical precision, 265Syncytium, 314Synthetic microtools fabrication

using direct laser writing, 90

TTaxis, 385T-cell stimulation, 398Teleoperation system, 202, 229Temperature-sensing microrobot, 279Temperature sensor

fabrication of, 279fluorescence images, 280

Temporal motion control, 416–417Test forces applied to probe, 366–369Thermal bonding process, 47Thermal microsources, 391

characterization, 391–392generation, 391–392

Thermotactic response cell migration, to local stimulation, 404to local thermal gradients, 400–403splitting of lamellipodium in response to, 402

Thermotaxis, 385, 386Thin film deposition, 302–303

bubble oscillation, 303–304microparticle targeting, 305–306optoelectronic tools, 304

Titanium-sapphire laser, 207T lymphocytes, 386T-matrix method, 6, 116Torque densities, 73Torque efficiency, 107, 119Tracking technologies, 3D, 21Transparent silica particles, 54Trapping configuration, schematic illustration,

69, 70Trapping effect, 130Trapping phenomenology, 73Traps containing nonspherical particles, 76–80

compound structures microtools, 79–80

trap stability criteria, 77–78trap stiffness, 76–77

Trypsinization, 287T-shaped nanorotor, 147Two-photon absorption (TPA), 254Two-photon photopolymerization (2PP), 34, 40, 82,

116, 118, 136, 169–172, 178applications, 118, 1193D direct writing by, 169microsubcomponents, HOT assembly, 51standard setup for structuring, 39

UUltrashort pulse lasers, 38Ultraviolet (UV)

fluorescence spectroscopy, 390lithography process, 83

Urethane acrylates, 40

VVaterite microspheres, 177

self-assembly of, 117Vector spherical wavefunctions (VSWF), 103Vertically trapped nanorod

nonconservative motion, 78Vibration-isolated tables, 10Video rate tracking, 16Virtual machine, 156Virtual micromachines, 156Viscous drag coefficient, 365Voxel size, as laser intensity function, 41VSWF. See Vector spherical wavefunctions (VSWF)

WWaveguide, nanowires. See Nanowires Wave-guided Optical Waveguides (WOWs), 271Whispering-gallery modes, 238

XX-box operating system, 20Xyz piezo positioner, 169

YYb fiber laser, 197Y-junction microchannel, 418

ZZeiss microscope, 19Zeolite L crystals, 419Zero-order beam, 198, 199