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8/12/2019 [AnnPhys 2013 Choi] Application of Femtosecond-pulsed Lasers for Direct Optical Manipulation of Biological Functio

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Ann. Phys. (Berlin), No. , () / DOI ./andp.

FEATURE ARTICLE

Application of femtosecond-pulsed lasers for direct optical

manipulation of biological functions

Jonghee Yoon1, Junseong Park1, Myunghwan Choi2,3 , Won Jong Choi1, and Chulhee Choi1,4,

Received 30 April 2012, revised 4 September 2012, accepted 11 September 2012

Published online 31 December 2012

Absorption o photon energy by cells or tissue can evoke

photothermal, photomechanical, and photochemical e-

ects, depending on the density o the deposited energy.

Photochemical effects require a low energy density and

can be used or reversible modulation o biological unc-

tions. Ultrashort-pulsed lasers have a high intensity due

to the short pulse duration, despite its low average energy.

Through nonlinear absorption, these lasers can deliver very

high peak energy into the submicrometer ocus area with-

out causing collateral damage. Absorbed energy delivered

by ultrashort-pulsed laser irradiation induces ree electrons,

which can be readily converted to reactive oxygen species

(ROS) and related ree radicals in the localized region. Free

radicals are best known to induce irreversible biological e-

ects via oxidative modication; however, they have also

been proposed to modulate biological unctions by releas-

ing calcium ions rom intracellular organelles. Calcium

can evoke variable biological effects in both excitable and

nonexcitable cell types. Controlled stimulation by ultrashort

laser pulses generate intracellular calcium waves that can

modulate many biological unctions, such as cardiomyocyte

beat rate, muscle contractility, and bloodbrain barrier (BBB)

permeability. This article presents optical methods that are

useul therapeutic and research tools in the biomedical eld

and discuss the possible mechanisms responsible or bio-

logical modulation by ultrashort-pulsed lasers, especially

emtosecond-pulsed lasers.

1. Introduction

Since the development of bright-field microscopy and

the first observation of cells in the 17th century by

Leeuwenhoek and Hooke [1], light has become an in-dispensable tool for biological research. A variety of

biomedical applications have used light to restore or

manipulate biological functions (Table 1) [2]. Light has

been applied in tumor treatment, a method known as

photodynamic therapy (PDT), by producing singlet oxy-

gen or free radicals that have toxic effects on tumor

cells or tumor-associated vasculatures [36]. Recently,

a new therapeutic modality, called low-level light ther-

apy (LLLT) has been developed and applied to regen-

erate wounds or alleviate pain through its photother-

mal or photochemical effects [79]. Lasers, especially

ultrashort-pulsed lasers, can disrupt the materials it con-tacts due to the photomechanical effects occurring at

high peak intensity. This property can be used for ma-

terial engineering, laser surgery such as laser-assisted in

situ keratomileusis (LASIK) or subcellular nanosurgery

[1014]. Optogenetics has recently emerged as a pow-

erful tool for studying cellular activities, and requires

photoactivatable receptors that react to light by chang-

ing their permeability; this facilitates manipulation of

the cellular functions of neurons or cardiomyocytes.

However, optogenetics has the drawback that it requires

genetic modification to produce photoactivatable re-

ceptors on target cells [1518]. A new optical methodhas been reported recently, in which ultrashort laser

pulses can be used to modulate various biological func-

tions without the need for genetic modification or exoge-

nous molecules [1924].

Corresponding author E-mail: [email protected] Department o Bio and Brain Engineering, KAIST, Daejeon, Korea2 Graduate School o Nanoscience and Technology, KAIST, Daejeon,

Korea3 Harvard Medical School and Wellman Center or Photomedicine,

Massachusetts General Hospital, 40 Blossom Street, Boston, Mas-

sachusetts 02114, USA4 KAIST Institute or the BioCentury, KAIST, Daejeon, Korea

C 2012 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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J. Yoon et al.: Application o emtosecond-pulsed lasers or direct optical manipulation o biological unctions

Table 1 Biomedical applications o light.

Field Mode of action Wavelength Energy Applications Remarks References

Low-level lasertherapy (LLLT)

PhotothermalPhotochemical

5001100 nm 14 J/cm2 - Wound healing Low backpain

[79]

PhotodynamicTherapy (PDT)

Photochemical(Singlet oxygen)

37 5732 nm 315 J/cm2 - Tumor treatment Need photosensitizer [36]

Laser ablation Photomechanical(Optical breakdown)

3551063 nm(ns,ps,s pulse laser)

1100 J/cm2 - Subsurace machining LASIK surgery Nanosurgery in biology

[1013]

Optogenetics Photochemical 450680 nm 213.8 mW/mm2 - Control cellular unctions(neurons, cardiomyocytes)

Need photoactivatablereceptor

[1517]

Multiphoton microscopy is a type of laser-scanning

microscopy that utilizes nonlinear effects of ultra-

short laser pulses. Most commonly, a near-infrared

femtosecond-pulsed laser is used as a primary source

due to its deep penetration, low scattering, and lo-

calized nonlinear absorption [25]. The probability of

nonlinear absorption is extremely small and propor-

tional to Ik (I = laser intensity, k = the number

of photons absorbed) [26, 27]. Thus, nonlinear mul-

tiphoton absorption occurs only on a tightly focused

region without out-of-focus fluorescence or phototoxi-

city [27]. These advantages allow nondestructive three-dimensional deep-tissue imaging even in highly scatter-

ing samples. Multiphoton microscopy is widely used for

biomedical research such as neuronal imaging [28], vas-

cular imaging [29], or in vivo investigations of tumor

physiology [30].

Ultrashort-pulsed lasers have been widely used

for manipulation of biomedical samples. The optical

tweezer technique is the method that manipulates nano-

to micrometer-sized particles in three spatial dimen-

sions by using forces generated by focused lasers [31].

Continuous-wave (CW) lasers are commonly used in the

optical tweezer technique; however, recently ultrashort-pulsed lasers, especially in the femtosecond range of

100 fs or less in pulse duration have been applied to

trap particles [32]. The combination of the multiphoton

imaging and optical trapping techniques proves to be a

valuable tool for biophotonics and cell study [33].

Ultrashort-pulsed lasers have also been used to ablate

intracellular organelle structures via laser-induced pro-

duction of low-density plasma [26,34]. Lastly, targeted ul-

trashort laser pulses have been used to modulate various

biological functions by controlling the intracellular Ca2+

concentration [1922, 24, 35]. Here, we briefly describe

the phenomenon of pulsed lasertissue interactions anddiscuss the possible mechanisms. We summarize our ob-

servations and current applications of the optical modu-

lation method using ultrashort-pulsed lasers, especially

femtosecond-pulsed lasers.

2. Direct optical modulation of biological

function using femtosecond-pulsed lasers

2.1. Lasertissue interaction

A highly focused pulsed laser induces multiphoton ab-

sorption, which can result in multiphoton ionization in

transparent materials, such as biological tissues or cells[3638]. The electron overcomes the bandgap energy and

becomes a free- or quasi-free electron via multiphoton

absorption (Fig. 1a). Once a quasi-free electron is pro-

duced, it obtains kinetic energy by absorbing photons,

a process known as inverse Bremsstrahlung (antibrak-

ing) absorption. When the kinetic energy of the excited

electron reaches the bandgap energy, it can ionize an-

other electron in the ground state by molecular colli-

sion, which is known as impact ionization. The recur-

ring sequence of inverse Bremsstrahlung absorption and

impact ionization leads to an ionization cascade, called

avalanche ionization (Fig. 1b) [26]. Multiphoton ab-sorption generates large numbers of quasi-free electrons

that can initiate avalanche ionization, and this process

generates a dense electron cloud, called a plasma. This

plasma generates cavitation bubbles, which produce a

rupture in the material due to violent mechanical effects

[26]. Therefore, this process is used mainly for ablative

applications, such as LASIK surgery [39].

The plasma formation process differs depending on

pulse duration and photon density. Nanosecond laser

pulses below the plasma formation threshold intensity

of 1011 W/cm2 do not produce free electrons. For the

production of seed electrons by multiphoton ionizationand subsequent avalanche ionization, irradiance val-

ues must reach the optical breakdown threshold value

C 2012 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheimwww.ann-phys.org


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Ann. Phys. (Berlin), No. ()

Figure 1 (online color at: www.ann-phys.org) Schematics o ultrashort-pulsed laser-induced plasma ormation. (a) Ground-state elec-

trons can overcome bandgap energy instantaneously by multiphoton absorption. Excited electrons that have sufficient kinetic energy

to escape rom local potential energy barriers are called quasi-ree electrons. (b) Quasi-ree electrons gain kinetic energ y via absorption

o the photon; this process is called inverse Bremsstrahlung absorption. Through the sequences o inverse Bremsstrahlung absorption,

quasi-ree electrons obtain sufficient energy and allow ground-state electrons o surrounding molecules to become new quasi-ree elec-

trons by transerring bandgap energy. This process is called impact ionization. Repeated inverse Bremsstrahlung absorption and impact

ionization amplies quasi-ree electron production and leads to ormation o a ree-electron cloud plasma.

for a nanosecond pulse. Nanosecond laser pulses at

the intensity of the over-irradiance threshold produce

too many electrons, which induces a rapid increase inavalanche ionization rate. Thus, free electrons gener-

ated by nanosecond laser pulses results in steep plasma

formation, which has a detrimental effect on biologi-

cal functions due to the high kinetic energy. Femtosec-

ond laser pulses generate plasmas with an intensity

of over 1013 W/cm2 in pure water. Unlike nanosecond

laser pulses, femtosecond laser pulses below the opti-

cal breakdown threshold can generate free electrons via

multiphoton ionization, which are not sufficient to ini-

tiate avalanche ionization. The density of free electrons

rises smoothly with increases in irradiance. Thus, free

electrons induced by femtosecond laser pulses below theoptical breakdown threshold value have a lower den-

sity than conventional plasma; this specific dense cloud

of free electrons is called low-density plasma. While

this low-density plasma has little destructive effect due

to its low kinetic energy, it can induce photochemical

effects that break chemical bonds or alter molecular

compositions [26].

2.2. Laser-induced plasma ormation and reactiveoxygen species (ROS)

Free electrons generated by laser irradiation induce ion-ization or dissociation of water and other molecules,

and subsequently produce reactive oxygen species

(ROS: superoxide, hydrogen peroxide, and hydroxyl

radicals) by electron delivery [40, 41]. Highly reac-

tive oxygen radicals induce oxidative modificationof cellular macromolecules, including proteins, lipids,

and DNA and cause irreversible damage and subse-

quent cell death. Oxidative modification of the var-

ious ion-transport proteins underlying ion channels

changes the permeability of channels and initiates ion

release [42].

The levels of intracellular ROS should be tightly reg-

ulated, and cells have developed strong antioxidant

defense systems to protect macromolecules from ox-

idative modification. The first ROS produced in mito-

chondria is the highly reactive superoxide (O2), which

superoxide dismutase (SOD) converts into a much morestable, and therefore relatively inert, ROS, hydrogen per-

oxide (H2O2). H2O2 can be further reduced to water

(H2O) by many antioxidant enzymes such as catalase,

peroxiredoxin (Prx), and glutathione peroxidase (Gpx)

[43, 44]. However, production of ROS in mitochondria is

accelerated by ROS themselves. Given oxidative stress,

ROS generation in only small numbers of mitochondria

can affect neighboring mitochondria, eventually propa-

gating an ROS surge throughout the cell via this positive

feedback loop [45, 46]. This phenomenon is called ROS-

induced ROS release (RIRR), and several studies have

revealed how loss of function in a small number of mito-chondria can influence overall cell functioning [4749].

Based on current knowledge, mitochondria-driven RIRR

C 2012 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ann-phys.org


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Figure 2 (online color at: www.ann-phys.org) Mechanism o ROS

propagation and ROS-induced calcium release. Laser-induced ROS

ormation in the ocal area inuences propagated ROS production

by the cellular mitochondrial network. Elevated intracellular ROS

induces calcium release into the cytosol rom the ER. This calcium

signal can be propagated to neighboring cells via gap junctions.

The yellow arrow indicates ultrashort-pulsed laser irradiation.

represents a mechanism of amplifying optically gener-

ated ROS (Fig. 2).

2.3. Irreversible effect o emtosecond-pulsed lasers onbiological samples

Laser pulses that have energy above the plasma for-

mation threshold induce submicrometer-sized bubbles

of plasma within a diffraction-limited volume [26]. For

the high-energy laser pulse, optical amplifiers are used

to increase the energy of each pulse while maintaining

the average energy, and thus, amplified femtosecond-

pulsed lasers show low repetition rates less than 10 kHz

[39]. This laser-induced plasma can precisely ablate

diffraction-limited volumes in tissue or specific or-

ganelles in the cell. Plasma-mediated ablation provides

the opportunity to study the role of specific biological

structures, including axons, microglial, mitochondria,

and microvessels by ablating withoutany significant heat

damage [39,50,51].

Below the plasma formation threshold energy, laser

pulses can also induce irreversible damage to the bi-

ological sample, especially in cells. Tirlapur et al. [40]

found that a mean power over 7 mW of unamplified

80-MHz 170-fs laser pulses generated ROS in scanned

regions. Laser-induced ROS resulted in impaired cell

division or initiated apoptotic cell death. Scanning

with low laser power and relatively long beam dwell

time (60120 s per pixel) induced a cytotoxic effect;

whereas brief exposures of high laser power with a short

beam dwell time (2 s per pixel) on a diffraction-

limited volume (femtoliter) in cells also evoked dam-

age [52]. This optical stimulation induced whole mi-tochondrial fragmentation even though the cytosolic

laser-exposed region was less than 1 m2 in area,

suggesting the involvement of the intermitochondria

network.

2.4. Optical modulation o various biological unctions

The cytoplasm is a restrictive medium for the diffu-

sion of charged compounds and ions such as ROS

because of its highly reducing environment. Thus, intra-cellular signaling systems utilizing ROS frequently oper-

ate via local communication between the sources and

targets [53, 54]. The endoplasmic reticulum (ER) can

be influenced by the ROS produced by mitochondria,

due to its close proximity to mitochondria and abun-

dance throughout the cytoplasm [55, 56]. Established

ROS-dependent regulators include Ca2+ channels (ryan-

odine receptors; RyRs and inositol 1,4,5-triphosphate re-

ceptors; InsP3Rs), cAMP-dependent kinases (PKA), and

Ca2+/calmodulin-dependent kinases (CaMK), that can

associate with Ca2+ transport proteins via anchoring

proteins (Fig. 2) [5760]. All of these scenarios suggestthat ER-mitochondrial coupling serve as the center stage

for ROS-Ca2+ cascade.

Recent biophotonic studies have indicated that

femtosecond-pulsed lasers stimulation could modulate

many biological functions, regardless of cell type by con-

trolling intracellular Ca2+ concentrations [20, 22, 25, 35].

Localized Ca2+ release from the ER through Ca2+ chan-

nels such as InsP3 or RyRs, initiates calcium-induced cal-

cium release (CICR) [61]. CICR, which is related to ER

Ca2+ release channels, plays a role in generation of in-

tracellular Ca2+ waves and is important in the excita-

tion of muscle cells and neurons [6264]. The intracel-

lular Ca2+ wave propagates to adjacent cells though gap

junctions, which are intercellular connections that allow

various molecules and ions to pass freely between cells

[65]. The processes mentioned above modulate Ca2+-

dependent signaling in tissues. Cells are classified into

two types: excitable cells that are able to produce and re-

spond to electrical signals, called action potentials; and

nonexcitable cells that also react to electrical signals,

but cannot produce action potentials. Although the ex-

citability of these cell types differs, the intracellular Ca2+

level of both is tightly regulated due to its physiological

importance [66, 67].



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2.4.1. Optical modulation of cellular functions in excitable cells

Excitable cell types including neurons, muscles, and se-cretory cells, require electrical signals to regulate their

functions. The difference in calcium ion concentration

between the inside and outside of the cell induces an

electrical potential, leading to changes in cellular activ-

ities. The entry of calcium ions into neurons causes ac-

tion potentials and neurotransmitter release. Ca2+ is also

an essential molecule for regulation of contraction of all

types of muscle [67].

Recently, optical methods of inducing action poten-

tials on neurons have emerged. One such method is use

of photoactivatable chemical molecules. Many chemical

compounds that could release bioactive molecules suchas glutamate or Ca2+ have been developed, and such

compounds are metaphorically termed caged com-

pounds or caged molecules [68]. The exposure of light

alters the chemical structure of caged compounds liber-

ating (uncaging) the caged bioactive molecule. Caged

compounds along with focusedlight irradiation could di-

rectly trigger membrane depolarization and action po-

tential in the neuron. However, the compounds should

be introduced into the neuron, and they have off-target

effects that limit functional specificity. Hence, caged

compounds are largely limited to in vivo applications

[69,70].Another optical method for manipulation of neuronal

activity using femtosecond-pulsed lasers has been de-

veloped. Hirase et al. [71] showed that exposure of an

76-MHz, 13-fs laser pulses of 780800 nm produced an

action potential on a pyramidal neuron in the absence

of any exogenous molecules like caged compounds. The

authors found that only mode-lock pulsed laser stim-

ulation could induce this action potential on the neu-

ron, but CW laser irradiation could not. This result in-

dicated that the nonlinear effect, especially multiphoton

absorption, is crucial to depolarization. Stimulation at

low intensity and for a longer exposure duration showeddifferent responses compared to a higher-intensity and

shorter-duration exposure. The former showed induc-

tion of sustained depolarization, which was mediated

by ROS, but the latter produced rapid depolarization,

which might be the result of membrane pore formation

due to photomechanical effects. Liu et al. [72] found that

femtosecond-pulsed laser stimulation triggered a cal-

cium wave in the irradiated hippocampal neuron. Then,

the laser-induced calcium wave propagated to adjacent

neurons, which allowed the authors to identify neural

circuits ex vivo. Because there was no need for an exoge-

nous probe or genetic modification, the femtosecond-

pulsed laser was proposed as a useful optical tool for

neurophysiology studies.

Femtosecond-pulsed laser stimulation can modulatemuscle contractility. There have been many reports that

femtosecond laser pulses generate intracellular cal-

cium waves in a variety of cell types [20, 24, 73]. By

controlling intracellular Ca2+ concentration,

femtosecond-pulsed laser stimulation could induce

muscle contraction, in which Ca2+ plays a critical role.

Muscle is classified into three types; skeletal, smooth,

and cardiac muscle. Skeletal muscle involves a voluntary

action that has a distinct series of alternating light and

dark bands perpendicular to the long axis. Skeletal

muscle fibers can be detected by label-free imaging

techniques, using autofluorescence or second-harmonicgeneration [74]. After laser stimulation, skeletal muscle

shows rapid twitch contraction and returns to its basal

length within several minutes (Fig. 3a). Smooth muscle,

located within the walls of blood vessels, the urinary

bladder, and respiratory tract, lacks the distinct banding

pattern found in skeletal muscle, and nerves innervat-

ing smooth muscle are derived from the autonomic

division. Thus, smooth muscle is not normally under

direct voluntary control. Femtosecond-pulsed laser

irradiation changes the cytosolic Ca2+ concentration,

leading to smooth muscle contraction without nerve

activity. This method can induce the contraction ofarterial blood vessels without use of exogenous probes,

such as caged molecules. Laser irradiation focused in the

brain artery wall caused localized circular contraction;

the artery recovered its basal lumen diameter within a

few minutes (Fig. 3b) [20]. Laser irradiation also caused

bladder smooth muscle contraction (Fig. 3c) [73]. The

bladder wall has a smooth muscle layer that controls its

capacity. Laser irradiation of dissected bladder smooth

muscle tissue induced localized increases in calcium

ion concentration, followed by whole smooth muscle

tissue contraction. The bladder smooth muscle fibers

recovered to their basal length within a few minutes.This optical method for modulation of muscle contrac-

tility can be used as an alternative therapeutic tool in

neuromuscular diseases.

Femtosecond laser pulses can alter the intracellular

Ca2+ concentrations in cardiomyocytes, a type of mus-

cle cell that provides contractility to the heart. Using an

82-MHz, 80-fs laser pulses of 780 nm, an 8-ms exposure

induced an intracellular Ca2+ wave, and the Ca2+ signal

propagated to nearby regions. Cardiomyocyte beat rate

was synchronized to laser irradiation frequency [23, 24].

Jenkins et al. [23] showed that a pulsed infrared diode

laser (= 1.875 m) coupled light into a multimode fiber



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Figure 3 (online color at: www.ann-phys.org) Optical modulation o contraction o different types o muscle. (a) Laser-induced skeletal

muscle contraction. Afer intravenous injection o 2 MDa FITC-dextran (green uorescence), the dorsal skinold chamber model in mice

was imaged with two-photon microscopy. Red uorescence indicates autouorescence o skeletal muscle bers under two-photon exci-

tation using a 760-nm Ti:Sapphire laser. The white dashed square in the baseline image indicates the region o laser irradiation. White

dashed lines indicate the baseline positions o capillaries. Yellow lines and white arrows indicate changes in capillary position caused

by skeletal muscle contraction. Scale bar, 50 m. (b) Laser-induced artery contractionin vivo. Green uorescence indicates the lumen o

blood vessels. The red dot and dashed line indicate the irradiated region and baseline vessel wall, respectively. Scale bar, 20 m. (c)Laser-

induced urinary bladder tissue contraction. Urinary bladder tissue was stained with the calcium indicator Fluo4-AM. Green uorescence

indicates theintracellularcalcium level o urinary bladder smoothmuscle bers. The reddot andwhite dashedline indicate theirradiatedregion and baseline position o smooth muscle bers, respectively. The white arrow and yellow line indicate changes in smooth muscle

ber length caused by smooth muscle contraction. Scale bar, 50 m.

400 m in diameter and modulated pacing of the em-

bryonic quail heart in vivo. This study used relatively

long (millisecond) pulses and a long wavelength, without

sufficient photon energy to overcome the bandgap en-

ergy. Thus, the laser intensity was insufficient to generate

low-density plasma directly. Although the mechanisms

remain unclear, both studies were remarkable in that

optical stimulation was shown to have the potential to

act as a pacemaker.

2.4.2. Optical modulation of cellular functions in nonexcitable

cells

The role of Ca2+ in nonexcitable cell types including ep-

ithelial cells, endothelial cells, and astrocytes, is differ-

ent from that in excitable cell types. Ca2+ signaling in

nonexcitable cells is closely involved in cell death, mi-

gration, and cell differentiation. Thus, nonexcitable cells

maintain low intracellular Ca2+ concentrations by tightly



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Figure 4 (online color at: www.ann-phys.org)

Optical modulation o BBB permeability.

(a) Time-series two-photon laser scanningmicroscopic images o a cortical vein in the

brain. Afer intravenous injection o 2 MDa

FITCdextran, the thinned-skull window was

imaged with two-photon microscopy. The red

dot indicates the region o laser irradiation.

Scale bar, 50 m. (b) Staining o astrocytes in

the brain using laser-induced extravasation.

Red uorescence indicates astrocytes stained

with the astrocyte specic dye SR101. Scale

bar, 50 m. (c) Local nuclear staining in the

brain cortex with Hoechst 33342. Image was

taken 30 min afer inductiono extravasation.Scale bar, 20 m.

regulating the flux of Ca2+ between cellular compart-

ments [66]. Despite their nonexcitability, femtosecond-

pulsed lasers can control the Ca2+ signaling and subse-

quent cellular functions of nonexcitable cells.

Femtosecond-pulsed laser stimulation can modu-

late bloodbrain barrier (BBB) permeabilityin vivo[19].

Brain microvascular endothelial cells are linked by tight

junctions that interconnect adjacent endothelial cells,

forming a physiological barrier, called the BBB. Most en-

dogenous and exogenous macromolecules do not cross

the blood vessel wall due to BBB. Thus, exogenous de-

livery of molecular probes or drugs is widely used for

in vivobrain research and brain-disease therapy. Recent

studies showed that unamplified 80-MHz, 120-fs pulsed

laser stimulation could modulate BBB permeability in

vivo. Brief laser exposure of the brain vein wall caused

a transient break in tight junctions and extravasation of

plasma into the brain parenchyma (Fig. 4a). We have ob-

served that the irradiated bloodvessel wall and BBB were

recovered within several minutes after stimulation [19].

By combining this method with systemic injection, laser-

induced extravasation can be used for local delivery of

functional molecular probes, such as the astrocyte stain-

ing dye SR101 (Fig. 4b), nuclear staining probe Hoechst

33342 (Fig. 4c), nanoparticles, and adenovirus, into the

brain. This optical method has the advantages of non-

invasive introduction of macromolecules into the brain

without opening the skull.

Astrocytes are the most abundant cell type in the

central nervous system (CNS). Unamplified 80-MHz

femtosecond-pulsed laser irradiation focused on a sin-

gle astrocyte induced intracellular calcium wave gen-

eration in the irradiated astrocyte in vitroand in vivo

[21, 35]. In response to elevation of intracellular calcium,

Figure5 (online color at: www.ann-phys.org) Vasodilationo the cerebral arteryby optical activationo surroundingastrocytes. Temporal

dynamics o astrocyte-mediated vasodilation. The dotted lines demarcate the arterial lumen at the baseline and the outer yellow linedemarcates the arterial lumen at 30 s. The white dot indicates the irradiated region. Scale bar, 10 m.



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astrocytes release neuromodulatory signaling molecules

that modulate vasomotion of the brain arteries. After

a short femtosecond-pulsed laser irradiation exposureof an astrocyte wrapped around an artery, the astro-

cyte showed rapid increases in levels of intracellular cal-

cium ions, followed by artery dilation in vivo (Fig. 5).

This optical method has drawbacks compared to other

techniques, such as caged molecules or optogenetics,

because it does not target specific molecular events.

However, it has the advantages of being label-free, non-

invasive, and does not show deleterious effects, such as

microglial activation.

3. Conclusion

Optical modulation of biological functions using

femtosecond-pulsed lasers has become an important

method in various biomedical fields. By allowing instan-

taneous high energy delivery to a three-dimensional

localized area, femtosecond-pulsed lasers can generate

low-density plasma. One effect of low-density plasma

is ROS production. ROS induce calcium ion release

through ER calcium channels, generating a calcium

wave that modulates many biological functions. Optical

approaches have advantages with regard to both preci-

sion and minimal invasiveness compared to chemicaland electrical methods. Optical methods for modu-

lating biological functions using femtosecond-pulsed

lasers provide new opportunities in areas ranging from

basic biological studies to the treatment of human

disease.

Ultrashort-pulsed lasers have many applications

other than modulation of biological functions described

above. These lasers can be applied to study functional

neural circuits by Ca2+ propagation, which is caused by

optical stimulation [75]. In addition, these lasers are uti-

lized in tumor treatment by targeting the vasculature

formation that results from tumor-associated aberrantangiogenesis. Laser irradiation generates a high dose of

ROS, which induces cytotoxic effects that result in de-

struction of the blood vessels that supply the nutrients

and oxygen required for tumor survival [76]. With the fur-

ther development of laser technology, the application of

ultrashort-pulsed lasers in biomedical fields will expand.

In particular, biological modulation methods combined

with imaging systems can be useful as both therapeutic

and research tools.

Acknowledgements. This research was supported by a grant(2011K000286) rom the Brain Research Center o the 21st Century

Frontier Research Program, unded by the Ministry o Education,

Science and Technology, the Republic o Korea (to C.C.).

Key words. Biophotonics, calcium, cell signaling, lighttissue in-

teraction, low-density plasma, optical modulation, photontissue

interaction, reactive oxygen species, ultrashort-pulsed lasers,

emtosecond-pulsed laser.

Chulhee Choi is Proessor and

Chair o theOptical Bioimaging

Center at Korea Advanced In-

stitute o Science and Technol-

ogy (KAIST). His researches are

ocused on developing in vivoimaging technique and sys-

tem, and discovering potential

drugable targets o malignantcancersusing in vivo-mimetic

tumor models. Recently, he is delineating the molecular

mechanisms o the tissue-photon interaction induced by

ultra-short pulsed lasers as a novel tool or modulation o

multiple cellular unctions.

Myunghwan Choi is a post-

doctoral research ellow at Har-

vard Medical School and Well-man Center or Photomedicine,

Massachusetts General Hospi-

tal. He has worked on in vivo

modulatory effect o ultrashort

pulsed lasers. He mainly con-

tributed on vascular permeability control, muscular con-

traction, and astrocyte activity control using ultrashort

pulsed lasers.

Junseong Park is a post-

doctoral research ellow atthe Inormation & Electronics

Research Institute o KAIST.

He has worked extensively on

many aspects o cell biology

and systems biology using

both wet work and compu-

tational methods. He mainly

contributed to elucidation o

cell signaling network and

progression o diseases including hepatitis C and cancer,

and identied many drug targets.



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Won Jong Choi received his B.S.

degree in biomedical engineer-

ing andappliedmathematics &

statisticsat Johns Hopkins Uni-

versity (Baltimore). Afer grad-

uation, he joined Cell Signaling

and Bio-Imaging lab at Korea Advanced Institute o Science

and Technology (KAIST). He studied the unctions o em-

tosecond laser and its relationship with calcium signaling

in muscle contraction.

Jonghee Yoonis a Ph.D. candi-

date in bio and brain engineer-

ing department at the Korea

Advanced Institute o Science

and Technology (KAIST). He

has studied biophotonics

using ultrashort-pulsed lasers.

He mainly contributed to

mechanisms o laser-induced

calcium wave generation and

applications or biomodu-

lation such as muscle contraction and cell death using

ultrashort-pulsed lasers.

References

[1] H. Gest, Notes Rec. R. Soc.58, 187201 (2004).[2] Q. Peng, A. Juzeniene, J. Chen, L. O. Svaasand, T. War-

loe, K.-E. Giercksky, and J. Moan, Rep. Prog. Phys. 71,056701 (2008).

[3] T. J. Dougherty, B. W. Henderson, C. J. Gomer, G. Jori,

D. Kessel, M. Korbelik, J. Moan, and Q. Peng, J. Natl.Cancer Inst.90, 889905 (1998).

[4] K. R. Weishaupt, C.J. Gomer, and T. J. Dougherty,Can-cer Res.36, 2326 (1976).

[5] C. J. Kelty, N. J. Brown, M. W. R. Reed, and R. Ackroyd,Photochem. Photobiol. Sci.1, 158168 (2002).

[6] D. W. Felsher, Nature Rev. Cancer3, 375380 (2003).[7] D. Hawkins, N. Houreld, and H. Abrahamse, Ann. N. Y.

Acad. Sci.1056, 486493 (2005).[8] R. T. Chow and L. Barnsley, Lasers Surg. Med.37, 46

52 (2005).[9] M. D. Skopin and S. C. Molitor, Photodermatol. Pho-

toimmunol. Photomed. 25, 7580 (2009).[10] B. Chichkov, C. Momma, S. Nolte, F. Von Alvensleben,

andA.Tunnermann, Appl. Phys. A Mater. Sci. 63, 109115 (1996).

[11] S. H. Chung and E. Mazur, Appl. Phys. A Mater. Sci.96,335341 (2009).

[12] H. Lubatschowski, G. Maatz, A. Heisterkamp, U. Het-zel, W. Drommer, H. Welling, and W. Ertmer, GraefesArch. Clin. Exp. Ophthalmol.238, 3339 (2000).

[13] S. Nolte, G. Kamlage, F. Korte, T. Bauer, T. Wagner, A.Ostendorf, C. Fallnich, and H. Welling, Adv. Eng. Mas-ter. 2, 2327 (2000).

[14] B. Girard, D. Yu, M. R. Armstrong, B. C. Wilson, C. M.Clokie, and R. J. Miller, Lasers Surg. Med. 39, 273285(2007).

[15] T. Bruegmann, D. Malan, M. Hesse, T. Beiert, C. J.Fuegemann, B. K. Fleischmann, and P. Sasse, NatureMethods 7, 897900 (2010).

[16] K. Deisseroth, Nature Methods8, 2629 (2011).[17] F. Zhang, M. Prigge, F. Beyriere, S. P. Tsunoda, J. Mat-

tis, O. Yizhar, P. Hegemann, and K. Deisseroth, NatureNeurosci. 11, 631633 (2008).

[18] L. Fenno, O. Yizhar, and K. Deisseroth, Annu. Rev.Neurosci. 34, 389412 (2011).

[19] M. Choi, T. Ku, K. Chong, J. Yoon, and C. Choi, PNAS108, 9256 (2011).

[20] M. Choi, J. Yoon, and C. Choi, J. Biomed. Opt. 15,015006 (2010).

[21] M. Choi, J. Yoon, T. Ku,K. Choi, and C. Choi, J. Biomed.Opt. 16, 075003 (2011).

[22] S. Iwanaga, T. Kaneko, K. Fujita, N. Smith, O. Naka-mura, T. Takamatsu, and S. Kawata, Cell Biochem. Bio-phys.45, 167176 (2006).

[23] M. W. Jenkins, A. R. Duke, S. Gu, Y. Doughman, H.Chiel, H. Fujioka, M. Watanabe, E. Jansen, and A.Rollins, Nature Photon. 4, 623626 (2010).

[24] N. I. Smith, Y. Kumamoto, S. Iwanaga, J. Ando,K. Fujita, and S. Kawata, Opt. Exp. 16, 86048616(2008).

[25] J. Ando, N. I. Smith, K. Fujita, and S. Kawata, Eur. Bio-phys. J.38, 255262 (2009).

[26] A. Vogel, J. Noack, G. Huttman, and G. Paltauf, ApplPhys B81, 10151047 (2005).

[27] W. R. Zipfel, R. M. Williams, and W. W. Webb, NatureBiotechnol.21, 13691377 (2003).

[28] J. N. Kerr and W. Denk, Nature Rev. Neurosci. 9, 195205 (2008).

[29] D. Kobat, M. E. Durst, N. Nishimura, A. W. Wong,C. B. Schaffer, and C. Xu, Opt. Exp. 17, 1335413364(2009).

[30] E. B. Brown, R. B. Campbell, Y. Tsuzuki, L. Xu, P.Carmeliet, D. Fukumura, and R. K. Jain, Nature Med.7, 864868 (2001).

[31] A. A. Ambardekar and Y. Li, Opt. Lett.30, 17971799(2005).

[32] J. C. Shane, M. Mazilu, W. M. Lee, and K. Dholakia,Opt. Exp.18, 75547568 (2010).

[33] J. Shane, M. Mazilu, W. M. Lee, and K. Dholakia, Proc.SPIE, 7038, 70380Y (2008).

[34] H. Sun, M. Han, M. H. Niemz, and J. F. Bille, LasersSurg. Med.39, 654658 (2007).

[35] Y. Zhao, Y. Zhang,X. Liu,X. Lv, W. Zhou, Q. Luo,and S.Zeng, Opt. Exp.17, 12911298 (2009).



10/10

Review

Article


[36] V. Venugopalan, A. Guerra III, K. Nahen, and A. Vogel,Phys. Rev. Lett. 88, 78103 (2002).

[37] W. Watanabe, N. Arakawa, S. Matsunaga, T. Higashi, K.Fukui, K. Isobe, and K. Itoh, Opt. Exp. 12, 42034213(2004).

[38] K. Konig, I. Riemann, P. Fischer, and K. Halbhuber,Cell. Mol. Biol. 45, 195 (1999).

[39] P. S. Tsai, P. Blinder, B. J. Migliori, J. Neev, Y. Jin, J. A.Squier, and D. Kleinfeld, Curr. Opin. Biotechnol. 20,9099 (2009).

[40] U. K. Tirlapur, K. Konig, C. Peuckert, R. Krieg, and K. J.Halbhuber, Exp. Cell Res.263, 8897 (2001).

[41] D. N. Nikogosyan, A. A. Oraevsky, and V. I. Rupasov,Chem. Phys.77, 131143 (1983).

[42] M.L. Circu and T. Y. Aw,Free Radic.Biol. Med. 48, 749762 (2010).

[43] I. Fridovich, Annu. Rev. Biochem. 64, 97112(1995).

[44] S. Orrenius, V. Gogvadze, and B. Zhivotovsky, Annu.Rev. Pharmacol. Toxicol.47, 143183 (2007).

[45] N. R. Brady, A. Hamacher-Brady, H. V. Westerhoff, andR. A. Gottlieb, Antioxid. Redox. Signal. 8, 16511665(2006).

[46] D. B. Zorov, C. R. Filburn, L. O. Klotz, J. L. Zweier, andS. J. Sollott, J. Exp. Med.192, 10011014 (2000).

[47] J. Park and C. Choi, Commun. Integr. Biol. 5, 8183(2012).

[48] J.Park, J.Lee, and C.Choi,PLoS ONE 6, e23211 (2011).[49] M. A. Aon, S. Cortassa, E. Marban, and B. ORourke, J.

Biol. Chem.278, 4473544744 (2003).[50] A. Nimmerjahn, F. Kirchhoff, and F. Helmchen, Sci-

ence 308, 13141318 (2005).[51] N. Nishimura, C. B. Schaffer, B. Friedman, P. S. Tsai, P.

D. Lyden, and D. Kleinfeld, Nature Methods 3, 99108(2006).

[52] J. Yoon, T. Ku, K. Chong, S. W. Ryu, and C. Choi, Proc.SPIE, 8221, 82210A (2012).

[53] G. Csordas and G. Hajnoczky, Biochim. Biophys. Acta1787, 13521362 (2009).

[54] R. Rizzuto and T. Pozzan, Physiol. Rev. 86, 369408(2006).

[55] G. Csordas, C. Renken, P. Varnai, L. Walter, D. Weaver,K. F. Buttle, T. Balla, C. A. Mannella, and G. Hajnoczky,

J. Cell Biol.174

, 915921 (2006).[56] C. Franzini-Armstrong, Physiology (Bethesda) 22,261268 (2007).

[57] M. J. Berridge, Cell Calcium32, 235249 (2002).[58] Y. J. Suzuki, and G. D. Ford, Am. J. Physiol.262, H114

116 (1992).[59] M. C. Camello-Almaraz, M. J. Pozo, M. P. Murphy, and

P. J. Camello, J. Cell. Physiol.206, 487494 (2006).[60] S. Zissimopoulos, N. Docrat, and F. A. Lai, J. Biol.

Chem.282, 69766983 (2007).[61] M. J. Berridge, M. D. Bootman, and H. L. Roderick, Na-

ture Rev. Mol. Cell Bio. 4, 517529 (2003).[62] A. Verkhratsky and A. Shmigol, Cell Calcium19, 114

(1996).

[63] M. W. Berchtold, H. Brinkmeier, and M. Muntener,Physiol. Rev.80, 12151265 (2000).

[64] M. J. Berridge, J. Physiol.586, 50475061 (2008).[65] D. A. Goodenough and D. L. Paul, Cold Spring Harb.

Perspect. Biol. 1, a002576 (2009).[66] A. C. Elliott, Cell Calcium30, 7393 (2001).[67] A. Burgen, Proc. R. Soc. Med.61, 67 (1968).[68] B. Judkewitz, A. Roth, and M. Hausser, Neuron 50,

180183 (2006).[69] R. H. Kramer, D. L. Fortin, and D. Trauner, Curr. Opin.

Neurobiol. 19, 544552 (2009).[70] A. Rana and R. E. Dolmetsch, Curr. Opin. Neurobiol.

20, 617622 (2010).[71] H. Hirase, V. Nikolenko, J. H. Goldberg, and R. Yuste, J.

Neurobiol. 51, 237247 (2002).[72] X. Liu, X. Lv, S. Zeng, W. Zhou, and Q. Luo, Appl. Phys.

Lett.94, 061113 (2009).[73] J. Yoon,M. Choi, and C. Choi, Proc. SPIE, 7897, 789714

(2011).[74] M. E. Llewellyn, R. P. Barretto, S. L. Delp, and M. J.

Schnitzer, Nature454, 784788 (2008).[75] V. Nikolenko, K. E. Poskanzer, and R. Yuste, Nature

Methods 4, 943950 (2007).[76] H. Choi, M. Choi, K. Choi, and C. Choi, Microvasc. Res.

82, 141146 (2011).


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