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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
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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
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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
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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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Ann. Phys. (Berlin), No. ()
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).
C 2012 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ann-phys.org
-
8/12/2019 [AnnPhys 2013 Choi] Application of Femtosecond-pulsed Lasers for Direct Optical Manipulation of Biological Functio
10/10
Review
Article
J. Yoon et al.: Application o emtosecond-pulsed lasers or direct optical manipulation o biological unctions
[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).
C 2012 by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheimwww.ann-phys.org