Introduction: Bio and Nano Imaging and Analysis

Research focusing on the intersection of life sciencesand nanotechnology is producing new fields such asnanobiotechnology, nanobiology and nanomedicine.This interaction is occurring in both directions. On onehand, biological molecules can be used to synthesizeand organize inorganic functional nanomaterials intowell-defined structures (Ma and others, 2008; Sotiro-poulou and others, 2008; van Bommel and others,2003). On the other hand, nanomaterials can be usedto study biology and develop biomedicines. For exam-ple, labeled nanomaterials can serve as effective long-lasting dyes for bio-imaging (Lee and others, 2007;Medintz and others, 2005; Xie and others, 2011) andnanomaterials are ideal drug-carriers for the develop-ment of new drug systems (Dobson, 2006; Nakanishiand others, 2009; Portney and Ozkan, 2006).

Microscopy is a powerful tool that can image bio-nanostructures, track nanomaterials in bio-systems,measure physical properties, determine compositions,and even create and manipulate nanostructures. Inthis special issue on bio-nano imaging and analysis,different microscopy techniques were used to charac-terize the structures of bionanomaterials, imagenanoparticles in biological systems, study biologicalfunctions, or manipulate nanostructures (Figure 1).This special issue includes 13 articles from leadingscientists in bionanotechnology and the microscopycommunity and covers the following four interestingtopics: 1) use of nanoparticles as imaging probes, 2)manipulation of nanostructures using microscopy, 3)bio-inorganic hybrid nanomaterials imaging, and 4)imaging nanomaterials using fluorescence andsingle-molecule microscopy.

In this special issue the first topic is about the use ofnanoparticles as imaging probes. Four frequently usednanoparticles and their principal method of imagingare summarized in table 1. This section includes threereview articles and one research article. The threereview articles concern recent advancements in theapplication of gold nanoparticles, magnetic nanopar-ticles and quantum dots for bio-imaging. The researcharticle presents a special method for labeling axonaltransport with quantum dots.

Subramanian Tamil Selvan et al, at the Institute ofMaterials Research and Engineering in Singaporehave reviewed the recent advances in the synthesisand application of bimodal magnetic-fluorescent probesfor bio-imaging (pages 563–576). Recent advances inimaging with nanoparticles, which include quantumdots, magnetic nanoparticles, rare-earth doped upcon-version fluorescent nanoparticles, and multifunctionalnanoparticles have been very rapid. These nanopar-ticles have not only enhanced imaging sensitivity,resolution, and specificity, but they have also allowedfor simultaneous multi-targeting, monitoring, and

enhanced diagnostics and delivery of therapeuticeffects. In the second part of this article, molecularimaging modalities clinically used such as positionemission tomography (PET), single photon emissioncomputed tomography (SPECT), magnetic resonanceimaging (MRI), optical and and ultrasound microscopyare summarized in a table along with the related imag-ing methods for the specific nanoparticles. Alsodescribed are recent advances in the assemblies of (1)superparamagnetic iron oxide (SPIO)-quantum dot(QD) based magnetic-fluorescent probes, (2) SPIO-rare-earth (RE) based magnetic-fluorescent probes, (3)Gd31-based magnetic contrast agents covalentlyattached to fluorescent probes, and (4) Gd31-basedMRI agents and fluorescent probes in a single nanoma-terial domain. This article should be important to thosestudying nanomaterials for bio-imaging.

The next article by Jesus Ruiz-Cabello from Univer-sidad Complutense de Madrid, Madrid, Spaindescribed use of magnetic iron oxide nanoparticles andgold nanoparticles (GNPs) in MRI imaging and genetherapy (Pages 577–591). The magnetic nanoparticlesare good contrast enhancement agents in MRI imag-ing. They can also be magnetically manipulated toguide delivery of genes into target cells for transfec-tion. When GNPs are modified with either Gd supra-molecular complexes or iron oxide, the new conjugatescan also serve as contrast enhancement agents in MRI.More importantly, they elucidate the conjugation ofthese nanoparticles with biological molecules such asviruses to form nanobioconjugates. This article sum-marizes how these nanobioconjugates can improve theperformance of the nanoparticles in MRI imaging andgene therapy. This article should be useful for thosewho are studying magnetic nanoparticles-based MRIimaging and gene delivery.

The article by Eliza Hutter and Dusica Maysinger,McGill University in Canada, reviews the unique prop-erties of GNPs and QDs, and how their properties ben-efit cellular and in vivo imaging (pages 592–604).GNPs have strong light scattering and surface plasmonenhanced luminescence, both of which can be used forbio-imaging. Light scattering by GNPs is usuallyvisualized by dark-field microscopy and surface plas-mon enhanced luminescence is most commonly moni-tored by two-photon luminescence microscopy. In thefirst part of this issue are described the physicochemi-cal characteristics of GNPs, how to apply GNPs in theimaging of cells and animals, advantages of applyingGNPs, and other potential applications of GNPs. QDs,which are stable, highly fluorescent, and tunable nano-particles, can be further functionalized for specificapplications. In the second part of this article, theauthors reviewed the use of QDs as bio-imaging probesto image protein location at cellular or the intracellularorganelle surfaces, to screen cancer markers in biologi-cal fluids, and to diagnose primary and metastatictumors in vivo. This article introduces two very usefulnanoparticles, luminescent QDs and plasmonic GNPs,

VVC 2011 WILEY-LISS, INC.

DOI 10.1002/jemt.21051Published online in Wiley Online Library (wileyonlinelibrary.com).

MICROSCOPY RESEARCH AND TECHNIQUE 74:559–562 (2011)

as bio-imaging agents. Researchers who are tryingto apply nanotechnology to develop biomedical imagingtechniques will find these nanoparticles particularlyhelpful.

The next research article is presented by BianxiaoCui et al, at Stanford University. They selected QDs asthe imaging probes for tracing axonal transport (pages605–613). This article presents a method of automaticimage analysis for constructing vesicle or cargo trajec-tories during axonal transport with higher data proc-essing throughput, better spatial resolution, and mini-mal axon perturbation. They first transformed theproblem of particle tracking in a 3D data set as a curvetracing problem in a 2D spatiotemporal kymographimage. They located the initial seeding points by identi-fying local intensity maxima within the kymographwhich were set as local brightest points. Then they car-ried out the core tracing algorithm using an implemen-tation of Steger’s algorithm, which is based on thedirection-dependence of the edge and intensityresponse functions between a template and the neigh-borhood of nearby pixels. To achieve high spatial reso-lution, they refined the position of each trajectory pointon the 2D kymograph image by back-projecting the tra-jectory points located in the kymograph onto the origi-nal image data and fitting the particle image with a 2DGaussian function. After all candidate segments wereextracted, they selected and connected the segmentsthrough an optimization. This article provides a new

method of automated data analysis for axonal trans-port processes.

The second topic, the manipulation of nanostruc-tures using microscopy, includes one review articlepresented by Jun Hu et al., Shanghai Institute ofApplied Physics in China. Hu reviewed recent find-ings on imaging and manipulation of biological mac-romolecular structures using atomic force microscopy(AFM) (pages 614–626). The authors highlighted thestudy of Ryanodine receptors and the dynamic pro-cess of peptide self-assembly by using the high reso-lution imaging capacity of the AFM. In the secondpart, they presented a method of cutting, pushing,isolating, and analyzing individual DNA moleculesby using AFM. In addition they introduced thevibrating mode scanning polarization force micros-copy (VSPFM), which could be used to study theelasticity of individual biomolecules and living cells.This article describes the AFM is a multi-faceted bio-physical tool for both imaging and nano-manipula-tion of biological structures on surfaces.

Five articles are included in the third topic of bio-inorganic hybrid nano-imaging. Because biomoleculeshave been frequently used as templates for the forma-tion of inorganic nanomaterials, imaging the resultantbio-inorganic hybrid nanomaterials is an extremelyimportant issue and is also a daunting challenge formicroscopists. These five articles are thereforeincluded in this special issue to highlight the progressmade in this research area. These articles cover severalmicroscopy techniques including negative staining,thin-sectioning, cryo-EM, advanced electron microsco-pies, and transmission X-ray microscopy.

The first article by Cao, Xu, and Mao at University ofOklahoma describes imaging bio-inorganic nano-hybrids by using transmission electron microscopy(TEM) as the primary tool (pages 627–635). TEM is apopular and relatively simple tool that can offer adirect visualization of the nanomaterials with highresolutions. When TEM is applied to visualize bio-inorganic nanohybrids, a treatment of negative stain-ing is necessary due to the presence of biological mole-cules in the nanohybrids. However, the conventionalnegative-staining procedure for regular biological sam-ples cannot be directly applied to such bio-inorganicnanohybrids. To image a specific bio-inorganic nanohy-brid, negative-staining factors such as negative staintype, working pH, staining time, and drying method,should be identified. Mao et al chose bacteriophage-gold nanoparticle hybrids as a model to systemicallystudy the effects of each factor on the negative stainingof the nanohybrids. They found the best staining condi-tions for imaging bacteriophage-gold nanoparticlehybrids and discussed the effects of each factor on thenegative staining of nanohybrids. This article shouldbe a helpful guide for scientists choosing the correct

TABLE 1. Nanoparticles as bio-imaging probes, their principal method of imaging, and equipment

Nanoparticles used as imaging probes Imaging Method Equipment

Gold nanoparticles Surface enhanced Raman spectroscopy Con-focal Raman microscopeQuantum dots Quantum confinements Fluorescence microcopeMagnetic nanoparticles Magnetic resonance imaging (MRI) MRI spectrometerUpconversion nanoparticles Upconversion luminescence Two-photon emission fluorescence microscope

Fig. 1. TheMicroscopy techniques used in this special issue to studybionanomaterials and bionanostructures. [Color figure can be viewed inthe online issue, which is available at wileyonlinelibrary.com.]

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negative staining conditions for imaging their bio-inor-ganic nanomaterials.

The next research article is presented by Qian Wanget al., at the University of South Carolina. The authorshave developed a method to characterize the proteindistribution in protein-polymer composites (pages 636–641). They assembled poly(4-vinylpyridine) (P4VP)with ferritin proteins into composite spheres and per-formed TEM thin-sectioning technique to investigatehow ferritin particles distributed within the spheres.In the process of thin-sectioning, the choice of fixativeand fixative infiltration are crucial steps. They foundthat when specimens were treated with an optimal pro-cedure for fixation and infiltration, a round-shapedthin sectioned structure with black and white regionswas observed. This article provides a new method toinvestigate the protein distribution in protein-polymercomposites which should be helpful for studying drugand gene delivery as well as developing new deliveryvehicles.

Bjoern Sander and Monika Golas from Aarhus Uni-versity in Denmark have reviewed TEM techniques forthe visualization of bionanostructures (pages 642–663).They describe the sample preparation techniquesincluding conventional negative staining, cryo-nega-tive staining, and unstained cryo-EM as well as thevarious imaging and image processing methods suchas electron crystallography, electron tomography, andsingle-particle EM. They also discussed how deviationsfrom an ideal symmetry and structural heterogeneitycan limit the resolution. This review article suggeststhat nanoimaging techniques, especially TEM, can notonly offer new applications in the field of nanomateri-als, but can also serve as powerful tools for the visual-ization and analysis of bionanostructures.

The next research article presented by Pratibha Gaiet al., from the University of York in United Kingdomdescribes the probing of nanostructures usingadvanced electron microscopy methods, including aber-ration-corrected transmission electron microscopy (AC-TEM), AC-high angle annular dark field scanningTEM (AC-HAADF-STEM), AC-energy filtered TEM,electron-stimulated energy dispersive spectroscopy inthe AC-(S)TEM and high-resolution TEM (HRTEM)with a scanning tunneling microscopy (STM) holder(pages 664–670). They used these techniques to imagethe presence of single Au atoms on titania surfaces incatalytic reactions, atomic-scale grain boundaries innanotwinned metals, atomically clean surface in nano-ZnO tetrapods, and crystallization and edge recon-struction in grapheme. These advanced electron mi-croscopy methods open up new opportunities for thestudy of nanomaterials at atomic scales and indicatefuture direction of electron microscopy development.

Joy Andrews et al., at SLAC National AcceleratorLaboratory in California introduced full-field transmis-sion X-ray microscopy (TXM) for nano-imaging of bio-materials (pages 671–681). Full-field TXM is a kind ofmicroscopy that can obtain a 3D view of both subcellu-lar and extensive intercellular structures in biologicalmaterials at the nano-scale. They first described theexperimental setup and sample preparation for TXM.Then different capabilities of TXM, including 3D to-mography, Zernike phase contrast, quantification ofabsorption, and chemical identification via X-ray fluo-

rescence and X-ray absorption near-edge structureimaging, were discussed and compared based on theresults from the imaging of biological materials such asmicroorganisms, bone, and plants. They discussed thecomplementarities of TXM with other imaging meth-ods.. This review article is informative to those who arenew to the field of TXM.

There are three articles in the fourth topic about flu-orescence and single-molecule microscopy. The firstresearch article is about using internal reflection fluo-rescence microscopy to study protein adsorption. Thesecond article compares AFM, cryo-TEM, and fluores-cence microscopy on imaging complicated biologicalnanostructures. The last one is about the study of pro-tein assemblies using tapping-mode AFM.

The first research article in this series by YanmeiWang et al. at Washington University in Missouriimaged the interactions of streptavidin moleculeswith hydrophobic fused-silica surfaces by single mol-ecule fluorescence imaging (pages 682–687). Con-trolled surface adsorption of proteins is importantfor protein-based sensors and protein microarrays. Itis necessary to identify and quantify mechanisms forprotein adsorption to surfaces. Wang et al used in-ternal reflection fluorescence microscopy to recordthe interaction of a single molecule, streptavidin-Cy3and streptavidin-Alexa555, with silica surfaces inreal time. They observed three different surfaceadsorptions which are deposition-process-associatedirreversible adsorption, reversible adsorption causedby direct interaction, and non-adsorption. Theresults indicate that both regulating postdepositionprotein-surface interactions and the deposition pro-cess should be taken into consideration in the studyof protein-surface adsorption.

In the next review article, Victoria Birkedal et al.from Aarhus University in Denmark investigated thecomplex self-assembled 3D DNA nanostructures byusing three single molecule microscopy techniques,which are AFM, cryogenic TEM, and fluorescence spec-troscopy and microscopy (pages 688–698). AFM imagedDNA self-assembled products (DNA sheets, openedDNA box, and closed DNA box) under liquid buffer con-ditions, and this method produced new informationabout the assembly and flexibility of the DNA box.Cryo-TEM showed unique insights into the structuraldetails of the 3D DNA box. FRET (Fluorescence Reso-nance Energy Transfer) spectroscopy provided valua-ble information on the lid opening process.The combination of several single molecule microscopytechniques generated more comprehensive informationand provided a detailed understanding of the archi-tecture, assembly, and functionality of biologicalnanostructures.

The last article presented by Jayne Garno et al. fromLouisiana State University is about the study of amy-loid peptide assemblies using tapping-mode AFM(pages 699–708). The aggregation of amyloid peptidesinto oligomeric and fiber assemblies can be a precursorof Alzheimer’s disease. The continuous monitoring ofsuch aggregation is important for a better understand-ing of the early accumulation of amyloid. They imagedthe assembly stages of fibril formation which includethe formation of seed nanoparticles, protofibrils, devel-opment of mature fibrils, densely branched network

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assemblies, and fibril bundles. This article demon-strates that characterizations using AFM arenecessary for tracking the evolution of biological nano-structures during time-dependent studies.

From these original research and review articles inthis special issue, it is evident that microscopy techni-ques have become one of the most important tools inthe characterization of bio-nanomaterials and eachtechnique has its unique advantages and disadvan-tages for imaging bio-, nano- or hybrid materials. It istherefore better for a researcher to know the principlesand applications of a variety of microscopy techniquesin this special issue and choose one or more of them fortheir specific needs. With the rapid development of mi-croscopy techniques for the imaging of bio-nanomateri-als, more and more functional bio-nanostructures willbe created for practical applications in the near future.At the same time, more novel functional nanomaterialswill be developed to serve as novel probes for research-ers to develop new microscopy techniques for bio-imag-ing. As a result, these developments will no doubtadvance the fields of nanobiotechnology, nanobiologyand nanomedicine.

ACKNOWLEDGMENTS

I am very grateful to all the contributors in this spe-cial issue. I would also like to thank Prof. GeorgeRuben who has given me encouragement and com-ments during the development of this special issue.Without their contributions, this special issue would beimpossible.

CHUANBIN MAO (Guest Editor)Department of Chemistry & BiochemistryStephenson Life Sciences Research Center

University of Oklahoma, Norman

REFERENCES

Dobson J. 2006. Gene therapy progress and prospects: magnetic nano-particle-based gene delivery. Gene Therapy 13:283–287.

Lee JH, Huh YM, Jun Y, Seo J, Jang J, Song HT, Kim S, Cho EJ, YoonHG, Suh JS, Cheon J. 2007. Artificially engineered magnetic nano-particles for ultra-sensitive molecular imaging. Nature Medicine13:95–99.

Ma N, Sargent EH, Kelley SO. 2008. Biotemplated nanostructures:directed assembly of electronic and optical materials usingnanoscale complementarity. Journal of Materials Chemistry18:954–964.

Medintz IL, Uyeda HT, Goldman ER, Mattoussi H. 2005. Quantumdot bioconjugates for imaging, labelling and sensing. Nature Mate-rials 4:435–446.

Nakanishi M, Inoh Y, Kitamoto D, Furuno T. 2009. Nano vectors witha biosurfactant for gene transfection and drug delivery. Journal ofDrug Delivery Science and Technology 19:165–169.

Portney NG, Ozkan M. 2006. Nano-oncology: drug delivery,imaging, and sensing. Analytical and Bioanalytical Chemistry384:620–630.

Sotiropoulou S, Sierra-Sastre Y, Mark SS, Batt CA. 2008.Biotemplated nanostructured materials. Chemistry of Materials20:821–834.

van Bommel KJC, Friggeri A, Shinkai S. 2003. Organic templates forthe generation of inorganic materials. Angewandte Chemie-International Edition 42:980–999.

Xie W, Qiu PH, Mao CB. 2011. Bio-imaging, detection and analysis byusing nanostructures as SERS substrates. Journal of MaterialsChemistry 21:5190–5202.

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