prospects and developments in cell and embryo laser nanosurgery

Overview

Prospects and developments incell and embryo laser nanosurgeryVikram Kohli∗ and Abdulhakem Y. Elezzabi1

Recently, there has been increasing interest in the application of femtosecond (fs)laser pulses to the study of cells, tissues and embryos. This review explores thedevelopments that have occurred within the last several years in the fields of celland embryo nanosurgery. Each of the individual studies presented in this reviewclearly demonstrates the nondestructiveness of fs laser pulses, which are used toalter both cellular and subcellular sites within simple cells and more complicatedmulticompartmental embryos. The ability to manipulate these model systemsnoninvasively makes applied fs laser pulses an invaluable tool for developmentalbiologists, geneticists, cryobiologists, and zoologists. We are beginning to seethe integration of this tool into life sciences, establishing its status amongmolecular and genetic cell manipulation methods. More importantly, severalstudies demonstrating the versatility of applied fs laser pulses have establishednew collaborations among physicists, engineers, and biologists with the commonintent of solving biological problems. 2008 John Wiley & Sons, Inc. Wiley Interdiscipl. Rev. Nanomed. Nanobiotechnol. 2009 1 11–25

Several studies have reported the application offemtosecond (fs) laser pulses as a precise scalpel

tool for performing cellular surgery.1–17 In each study,fs laser pulses were produced from a titanium sapphire(Ti:Sapphire) laser oscillator or amplifier (700–900nm) delivering a sub-10 fs to 250 fs pulse at arepetition rate of 76 MHz to 1 kHz. The fs laserpulses were coupled to a high numerical aperture (NA)microscope objective, NA = 0.95–1.4, and localizedto cellular and subcellular sites. Beam dwell timesranged from milliseconds to seconds and pulseenergies delivered to the sample were 0.03 to severalnanojoules per pulse (nJ/pulse). Model systems thathave been used in fs laser pulse mediated nanosurgeryinclude human metaphase chromosomes,4 Chinesehamster and canine kidney epithelial cells,1,2 plantchloroplasts,5 mitochondria in endothelial and HeLacells,6,7 yeast microtubules,8 the actin cytoskeletonin fixed 3T3 fibroblast and bovine endothelialcells,6,9 hamster ovary cells,10,17 Caenorhabditiselegans,11,12 Drosophila melanogaster,16 Sprague-Dawley rats and Danio rerio (zebrafish).13 Using these

∗Correspondence to: Vikram Kohli, University of Alberta, Edmon-ton, Alberta, Canada.E-mail: [email protected] of Electrical and Computer Engineering, University ofAlberta, Edmonton, Alberta, Canada

DOI: 10.1002/wnan.029

biological systems, intrachromosonal dissections,4

membrane surgery,1 cell isolation,1 cytoskeletal andmicrotubule ablation,6,8,9 knockdown of plastids,5

laser axotomy of neurons,11 intravascular disruptionof microvessels,13 cellular delivery of exogenous DNA,carbohydrates and quantum dots2,3,17 and the surgicalablation of Drosophila16 and zebrafish embryos3,15

have been demonstrated. In this paper, we present areview of current developments in fs laser mediatednanosurgery of cells and embryos with emphasis onthe fs laser as a tool able to induce ablation withhigh spatial resolution and with minimal transferof thermal and mechanical stresses to the materialinvestigated.

LASER INTERACTION WITHBIOLOGICAL MATERIALSFeatures that distinguish fs laser pulses from longerpulse durations (i.e., nanosecond pulses) include theability to localize cellular disruption to a sub-micronresolution, the low threshold energy needed to elicitablation and the lower conversion of energy intoshockwaves and cavitation bubbles, which are adverseside effects known to increase the spatial extentof cellular damage.18–22 When fs laser pulses arefocused to a high peak intensity of 1011–1013 W/cm2,optical breakdown occurs, resulting in the ablation of

Volume 1, January /February 2009 2008 John Wi ley & Sons, Inc. 11

Overview www.wiley.com/wires/nanomed

the biological material.19 The mechanism by whichthe material is ablated depends on the strength ofthe peak intensity, leading to quasi-ionized electrons(the electrons are not completely ionized from theatom, rather they occupy a higher energy state withinthe conduction band of the material) produced viamultiphoton absorption or tunneling ionization.19

The Keldysh parameter19,23,24

γ = ω

e

√cε0m�

4I(1)

determines the extent to which multiphotonabsorption or tunneling ionization governs the abla-tion process. In Eq. (1), ω, e, c, ε0, m, �, I representthe frequency of light, electron charge, speed of light,permittivity of free space, electron-hole reduced mass,bandgap energy, and peak intensity of the light pulse,respectively. When γ < 1, tunneling ionization dom-inates the laser-matter interaction process, while forγ > 1 multiphoton ionization dominates.19,23 As to avery good approximation we can consider biologicaltissue as water, Sacchi25 proposed that water shouldbe modeled with a bandgap energy of 6.5 eV. Thevalue of 6.5 eV arises from work conducted by Boyleet al.26 which examined the photolysis of liquid water.If we consider laser light produced from a Ti:Sapphirelaser oscillator with an emission spectrum centered at800 nm (1.55 eV) and a pulse duration of 100 fs, thenfor γ > 1, the biological tissue must simultaneouslyabsorb five photons to excite a valence electron to theconduction band. This conduction electron representsa ‘seed electron’ that undergoes free carrier linearabsorption by nonresonantly absorbing laser photonsthrough inverse bremsstrahlung.19,23 As the electronenergy increases, a condition is reached where theelectron undergoes impact ionization,19 defined as1.5�̃ 24 where �̃ is the effective ionization potentialgiven by19,24

�̃ = 2π

�

√1 + γ 2

γE

(π

2, k

)(2)

where E(

π2 , k

)represents the elliptical integral of the

second kind with k = (1 + γ 2)−1/2.19 At 1.5�̃ the elec-tron impact ionizes a valence electron, resulting in twoelectrons in the conduction band (seed electron andionized valence electron). Through linear absorptionof laser photons, these two electrons can participatein impact ionization, causing a rise in the densityof conduction band electrons. This cascade effectis properly termed avalanche ionization, where theionized electron density quickly rises to a critical valuewhere optical breakdown occurs.19,20,23 At optical

breakdown, the plasma frequency equals the laserfrequency and the critical electron density becomes20

Ncrit = ω2meε0

e2 (3)

where me is defined as the electron mass. At awavelength of 800 nm, Ncrit = 1021 cm−3, beyondwhich the plasma becomes highly reflective andabsorbing to laser light.19

As a consequence of using fs laser pulses forablation, seed electrons can be generated with anintensity value lower than the threshold intensityfor optical breakdown.19 With nanosecond laserpulses, no seed electrons are created by multiphotonionization for intensities below the threshold foroptical breakdown.19 Therefore, the requirement thatthe intensity must equal the threshold for opticalbreakdown to produce seed electrons indicates anincrease in the deposition of laser energy. However,this increased energy is funneled into shockwaves andcavitation bubbles, leading to a larger spatial disrup-tion of the material. In fact, Vogel et al. showed thatthe conversion of energy into cavitation bubbles for fslaser pulses was 6.8% versus 12.7% for nanosecondpulses.19 As a result, less energy is required to elicitablation of the material, which reduces the amount oftransient stresses such as shockwaves and cavitationbubble formation imparted to the sample.19

In fs laser-tissue interaction the mechanism ofablation between high and low repetition rate laseroscillators (i.e., 80 MHz vs. 1 kHz) is different.19

For instance, in 80 MHz ablation the pulse energyis below the threshold energy for optical breakdownwith each pulse producing a low density plasma.19

Ablation of the biological material occurs through theinteraction of multiple pulses through free electroninduced chemical decomposition of the material viabond-breaking.19 In contrast, with low repetition ratessuch as 1 kHz, the pulse energy for ablation is near orabove the breakdown threshold energy. Larger plasmadensities are created in comparison to 80 MHz withthe formation of minute cavitation bubbles. It has beensuggested that the cavitation bubbles are responsiblefor the dissection of the biological material.19

Using fs laser pulses, highly spatially localizedablation is achievable through the nonlinear multipho-ton ionization process (γ > 1). When a laser beam isfocused by a high NA objective, it is the electrondensity profile and not the irradiance profile that gov-erns the spatial extent of ablation. (The NA of themicroscope objective lens can alter the ablation pro-file. Using low NA objectives (i.e., NA < 0.9) plasmascan be generated ahead of the focus. Such a plasma

12 2008 John Wi ley & Sons, Inc. Volume 1, January /February 2009

WIREs Nanomedicine and Nanobiotechnology Developments in cell and embryo laser nanosurgery

can effectively shield successive laser photons fromreaching the focus and induce plasma defocusing.19,27

Spatially asymmetric plasmas are created with lowNA objectives, with the observance of a high plasmadensity (before the geometrical focus) surrounded bya lower density region.27 In contrast, for NA ≥ 0.9,smaller symmetric plasmas are formed.27) The diffrac-tion limited laser spot size has transverse, dtrans, andlongitudinal, z, dimensions28

dtrans = 1.22λ

NAand z ≈ 2

(πw2

0

λ

)(4)

where, for example, NA = 1.3, λ = 800 nm and w0 =375 nm are the NA, wavelength of light and radius ofthe beam waist, respectively. The irradiance profilesalong the transverse and longitudinal direction are750 and 1104 nm, respectively. As the simultaneousabsorption of five photons is required to produce aseed electron, the ablation dimensions are effectivelyreduced by

√5,19 yielding 335 and 494 nm for the

transverse and longitudinal dimensions, respectively.(The transverse and longitudinal dimensions of theablation profile represent theoretical estimates. Theformation of cavitation bubbles, particularly withhigh repetition rate laser oscillators (i.e., MHz), canincrease these values.) Therefore, the ablation ofbiological tissue can be localized to a high spatialresolution, allowing key structures within biologicalmaterial to be removed or altered without affectingadjacent cellular sites. This unique property has madethe application of fs laser pulses a novel tool for thenondestructive study of biological materials.

Cellular and Subcellular NanosurgeryIn a study by Konig et al.,4 the authors reported thenanodissection of fixed air-dried human metaphasechromosomes using fs laser pulses (170-fs, 800 nm,80 MHz). Intrachromosonal dissections were madeby 500–2500 consecutive single line scans across thechromosome using an average laser power of 100mW (1.25 nJ/pulse).4 Dissection depths were analyzedusing scanning force microscopy and revealed a fullwidth at half maximum cut size of 170 ± 10 nm for500 consecutive scans.4 A reduction in the numberof laser line scans to 250 produced smaller cut sizeson the order of 85 ± 10 nm.4 It was also found thatthe cut sizes increased with an increasing number ofconsecutive scans, from 200 to 400 nm for 1000 to2500 scans. In addition to line scans, the authorsperformed stationary ablation of the chromosomeswith an average laser power of 15 mW (0.19 nJ/pulse)and varying beam dwell times.4

(a)

(c)

(b)

FIGURE 1 | Membrane surgery on a live MDCK cell. (a) Illustrates acell of 12 µm in length where three ∼ 800 nm incisions have beenmade. (b) When the sample is traversed along its long axis anadditional incision is made, with (c), two extra sub-micron surgicalincisions. The arrows in (a) indicate the ablated extracellular matrixsecreted by the cell. Unlike fibroblasts, MDCK cells are devoid of focaladhesions, and cell-substrate bonds anchor the cell to the substrate.With precise sample movement, the isolation of single MDCK cells canbe achieved when the laser traces the exterior contour of the cellmembrane. Laser parameters: pulse duration sub-10 fs; excitationwavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy forsurgery 5 nJ/pulse; 0.95 NA 100× air microscope objective. (Reprinted,with permission, from Ref. 1. Copyright 2005 Wiley Periodicals, Inc.).

In the work conducted in our lab, Kohli et al.1

demonstrated the surgical dissection of biologicalmaterial using fs laser pulses. With a pulse energyof 5 nJ/pulse (sub-10 fs, 800 nm, 80 MHz)several dissection cuts were made in the plasmamembrane of live Madin-Darby Canine Kidney(MDCK) cells. 1 Figure 1 shows membrane surgery onthe mammalian cell, where the arrows represent theablated extracellular matrix. Post-laser surgery, thecell maintained normal morphology without evidenceof membrane re-orientation, cell collapse or blebformation, Figure 1. It was hypothesized that theabsence of cell disassociation after laser surgery waslikely as a result of coalescence of the dissected upperand lower plasma membrane.1 Further work by thisgroup used fs laser pulses as a novel tool for single cellisolation. Figure 2 depicts nanosurgical isolation of alive Chinese hamster fibroblast cell. Two fibroblastcells are shown initially tethered together by a focaladhesion. As shown in Figure 2(b–d), by scanning thecells along the dissection interface relative to the laser



(a) (b)

(d)(c)

FIGURE 2 | Live video observation of nanosurgical isolation of livefibroblast cells. (a) The arrows depict two fibroblast cells (V79-4), with atethered width of ∼1 µm. The dashed line represents the dissectioninterface the sample traverses relative to the fs laser spot. (b) Theapplication of focused laser pulses (1013 W/cm2/pulse), indicated by thearrow, nanosurgically ablates the focal adhesions adjoining the twofibroblast cells. (c) The surgery precisely isolates and detaches the cell,indicated by the dotted box. This is achieved without morphologicallycompromising the cell. (d) An in-focus image, depicted in the dottedbox, shows a live isolated folded fibroblast cell. Laser parameters: pulseduration sub-10 fs; excitation wavelength 800 nm; oscillator repetitionrate 80 MHz; pulse energy for surgery 5 nJ/pulse; 0.95 NA 100× airmicroscope objective. (Reprinted, with permission, from Ref. 1.Copyright 2005 Wiley Periodicals, Inc.).

spot, removal of the focal adhesion resulted in theisolation of a single fibroblast cell from its neighbor.1

Shen et al.6 ablated fluorescently labeled actincytoskeleton in fixed 3T3 fibroblast cells using apulse energy ranging from 1.5 to 3 nJ/pulse (100-fs, 1 kHz). By translating the cells relative to thelaser pulse, nanometer scale channels were made inthe cytoskeleton. The diameter of the ablated channelswas found to decrease as the pulse energy was lowered,with a threshold for actin ablation of 1.5 nJ/pulse.6

Confirmation that the cytoskeleton was ablated andnot photobleached was obtained by restaining the cellsafter laser irradiation.

Heisterkamp et al.9 also dissected fluorescentlylabeled actin in both fixed and live bovine capillaryendothelial cells using a pulse energy ranging from1.8 to 4.4 nJ/pulse (100-fs, 1 kHz). Similar to the

(a)

(b)

(5 µm)

0 s 2 s 5 s

FIGURE 3 | (a) Fluorescence microscope image of GFP-labeledmicrotubule network in an endothelial cell. (b) Time-lapse sequenceshowing rapid retraction of microtubule due to depolymerization. Thecross hair shows the position targeted by the laser; the arrows show theretracting ends of the microtubule. Laser parameters: pulse duration200–250-fs; excitation wavelength 790 nm; oscillator repetition rate 80MHz; pulse energy for dissection of GFP-labeled microtubule 1.5–1.8nJ/pulse; 1.4 NA oil immersion microscope objective. (Reprinted, withpermission, from Ref. 9. Copyright 2005 Optical Society of America).

observations of Shen et al., the width of the cuts wasfound to decrease as the pulse energy was reduced,from 600 to 240 nm for 4.4 and 2.2 nJ/pulse,respectively.9 Figure 3 shows the dissection of a singlegreen fluorescent protein (GFP) tagged microtubule ina live cell irradiated with 1000 pulses at a pulse energyof 1.5 nJ/pulse. It was found that within 2 s, the micro-tubule retracted because of depolymerization9 (Figure3(b)) (arrows). In addition to the ablation of actin,the authors also dissected the nucleus in a fixedendothelial cell. Transmission electron microscopyanalysis revealed that the nucleus could be ablatedwith a pulse energy as low as 1.8 nJ/pulse.9

Recently, Sacconi et al.8 demonstrated nano-surgery (100-fs, 80 MHz) of GFP-labeled micro-tubules in fission yeast cells. Individual mitotic spin-dles in anaphase B were irradiated with an averagelaser power of 4 mW (0.05 nJ/pulse) for 150 ms.8



(a)

(b)

(c)

(d)

0 255

FIGURE 4 | Emboss-filtered transmission (a),(c) andpseudo-color-coded autofluorescence images (b),(d) of chloroplasts inthe epidermal cell of E. densa before (a),(b) and 8 s after (c),(d) selectiveknock-out of part of a specific chloroplast (lightning symbol) with 800nm near infrared (NIR) fs laser pulses at a mean power of 30–50 mW inthe presence of the cell-impermeate fluorescent dye propidium iodide(PI). Note the active movement of the chloroplasts in the corticalcytoplasmic region of the target cell (arrowheads) as well as in theadjacent cells (arrows) after nanoprocessing. No PI fluorescence isdiscernible in the cytoplasm of the cells, indicating that the cells remainviable. Distinct cytoplasmic streaming in the cortical region of the cellswas invariably present even after 30 min. Scale bar = 50 µm. The insetpseudo-color-coded bar represents a pixel intensity profile between 0and 255 units. Laser parameters: pulse duration 170-fs; excitationwavelength 720 nm; oscillator repetition rate 80 MHz; pulse energy fornanodissection of chlorplast 0.38–0.63 nJ/pulse; beam dwell time forirradiation 13 ms. (Reprinted, with permission, from Ref. 5. Copyright2002 Blackwell Publishing).

Following nanosurgery, the spindles were bent andbroken into segments. In a similar experiment, theauthors determined the optimal average laser powerfor nanosurgery of cytoplasmic microtubules in inter-phase cells. For an average laser power below 2 mW(0.03 nJ/pulse),8 it was shown that the shape andlength of the microtubule remained unchanged afternanosurgery. However, above 2 mW, disassociationof the microtubules was observed, with the frequencyof breakage increasing with higher average laser pow-ers (4–8 mW). The optimal average laser power formicrotubule disassociation was found to be 4 mW,

20 µm(a) (b)

(c)

FIGURE 5 | Ablation of a single mitochondrion in a living cell.(a) Fluorescence microscopic image showing multiple mitochondriabefore fs laser irradiation. Target mitochondrion (marked by arrow)(b) before (c) after laser ablation with 2 nJ pulses. Laser parameters:pulse duration 100-fs; oscillator repetition rate 1 kHz; excitationwavelength 800 nm; pulse energy for the irradiation of mitochrondrion2 nJ/pulse; 1.4 NA oil immersion microscope objective. (Reprinted, withpermission, from Ref. 6. Copyright 2005 Tech Science Press).

yielding a 75% disassociation efficiency and 100%cell survival.8

Konig et al.10 demonstrated the nanodissectionof chromosomes in the nucleus of live Chinese hamsterovary cells, using a pulse energy of 0.4 nJ/pulse andan exposure time of 500 µs. At this pulse energy, chro-mosomes could be ablated without disruption to thenuclear envelope. However, at 0.63 nJ/pulse, dissec-tion of the chromosomes was accompanied by damageto the nuclear envelope and the outer cell membrane.10

In a study by Tirlapur and Konig,5 the authorsused fs laser pulses (170-fs, 720 nm, 80 MHz) for thenanodissection of plant cell walls and the partial andcomplete removal of chloroplasts in Elodea densa.Using an average laser power ranging from 30 to50 mW (0.38–0.63 nJ/pulse), lesions with a widthof< 400 nm were made in the plant cell wall.5 Figure4 depicts transmission and autofluorescence images ofthe chloroplast in E. densa before and after removalof this organelle. Figure 4(a, b) shows several chloro-plasts in the epidermal cell of the plant (arrows), wherethe lightning symbol identifies the chloroplast chosenfor removal. Using an average laser power of 30 mWand a beam dwell time of 13 ms, portions of thetargeted chloroplast were removed, (Figure 4(c, d)),without compromising the functionality or integrityof adjacent chloroplasts.5 To verify that adjacent plas-tids remained functional, phase-contrast transmissionmicroscopy was used to examine the cytoplasmicmovement of the organelles in the cortical region.Normal cytoplasmic movement was observed in allnonirradiated chloroplasts.5 To address whether sub-cellular removal of the plastids altered cell viability, anexamination of the presence of propidium iodide (PI)



0.60.50.40.4

0.5

0.6

0.7

0.8

0.9

1.0

0.3Time (s)

V/V

equi

l

0.20.10.0

(a) (b) (c)

FIGURE 6 | The response of a micropatterned MDCK cell suspended in 1.0 M sucrose when permeabilized by fs laser pulses. (a) MDCK cell beforepermeabilization. The arrow depicts the focused fs laser spot. Only one cell was chosen for permeabilization, demonstrating the precision of theprocess. (b) MDCK cell after permeabilization. The cell has increased in cellular size towards equilibrium volume. The arrow in (b) illustrates thepermeabilized cell. (c) Volumetric response of a micropatterned MDCK cell in a 0.2 M cryoprotectant sucrose solution. Initially the cell is in ashrunken state. Upon laser permeabilization, the cell quickly swells to equilibrium volume. The value of Vequil was taken to be the equilibriumvolume as measured using Image J analysis software. Scale bar in (a) and (b) is 40 µm. Laser parameters: pulse duration sub-10 fs; excitationwavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy for permeabilization 3 nJ/pulse; 0.95 NA 100× air microscope objective.(Reprinted, with permission, from Ref. 2. Copyright 2005 Wiley Periodicals, Inc.).

Before

(a) (b) (c) (d) (e)

After 6 h 12 h 24 h

FIGURE 7 | Fs laser axotomy in Caenorhabditis elegans worms using 100 pulses of low energy (40 njJ) and short duration (200 fs) and a repetitionrate of 1 kHz. Fluorescence images of axons labeled with green fluorescent protein before, immediately after, and in the hours following axotomy.Arrow indicates point of severance. Scale bar, 5 µm. Laser parameters: pulse duration 200-fs; oscillator repetition rate 1 kHz; pulse energy for laseraxotomy 40 nJ/pulse; 1.4 NA 64× oil immersion microscope objective. (Reprinted, with permission, from Ref. 11. Copyright 2004 Nature PublishingGroup).

in the cytoplasm of the irradiated cells was performed.Using transmission and two-photon fluorescence, noaccumulation of PI was observed in the targeted cells,indicating that the cells remained viable.5

Shen et al.6 targeted a single fluorescentlylabeled mitochondrion in bovine adrenal capillaryendothelial cells with fs laser pulses (100-fs, 1 kHz).The purpose of the study was to elucidate theconnective properties of mitochondria to determinewhether this organelle forms a continuous networkor represents an independent structural unit. Afterstationary irradiation of the mitochondrion with afew hundred pulses at an energy of 2 nJ/pulse,surgical removal of the mitochondrion from theendothelial cell was accomplished without affectingneighboring mitochondria6 (Figure 5). Since only the

targeted mitochondrion was structurally damaged andremoved, (Figure 5(b,c) arrow), the authors claimedthat the absence of adjacent mitochondrial damageprovided direct evidence that this organelle exists asan independent unit.6

Watanabe et al.7 removed a mitochondrion ina human carcinoma cell line, HeLa, using a pulseenergy ranging from 2 to 7 nJ/pulse (150-fs, 1 kHz)and a beam dwell time of 250 ms. At 7 nJ/pulse,the removal of the mitochondrion was accompaniedby plasma membrane disruption indicated by cellularPI uptake.7 However, membrane disruption was notobserved following mitochondrial ablation for pulseenergies between 2 and 4 nJ/pulse, indicating thatthe cells remained viable after laser irradiation. Theauthors used confocal imaging to confirm that the



Hemorrhage

Intravascularclot

Extravasation

FIGURE 8 | Schematic of the three different vascularlesions that are produced by varying the energy and numberof laser pulses. At high energies, photodisruption produceshemorrhages, in which the target vessel is ruptured, bloodinvades the brain tissue, and a mass of red blood cells(RBCs) form a hemorrhagic core. At low energies, the targetvessel remains intact, but transiently leaks blood plasmaand RBCs forming an extravasation. Multiple pulses at lowenergy lead to thrombosis that can completely occlude thetarget vessel, forming an intravascular clot. Scale bars,50 µm. Laser parameters: pulse duration 100-fs; oscillatorrepetition rate 1 kHz; pulse energy for inducing vascularlesions 0.03–0.50 µJ/pulse; 0.8 NA 40× water immersionmicroscope objective. (Reprinted, with permission, fromRef. 13. Copyright 2006 Nature Publishing Group).

targeted mitochondrion was ablated, and that itsabsence (as detected by fluorescence microscopy) wasnot because of its diffusion out of the focal plane (bycytoplasmic streaming).7 Similar to the observationsof Shen et al.,6 Watanabe reported that neighboringmitochondria remained intact.

In a study by Tirlapur and Konig,14 the authorsemployed fs laser pulses (800 nm, 80 MHz) tointroduce DNA into Chinese hamster ovarian (CHO)cells and rat-kangaroo kidney epithelial cells (PtK2).With an average laser power ranging from 50 to 100mW (0.625–1.25 nJ/pulse), the cell membrane wasdisrupted in the presence of DNA plasmid vectorpEGFP-N1 encoding enhanced green fluorescentprotein (GFP). Disruption of the cell membrane after16 ms of irradiation resulted in the introduction ofDNA.14 Expression of the DNA construct was verifiedby two-photon fluorescence imaging.14

In similar work to that of Tirlapur and Konig,Stevenson et al.17 transfected CHO cells with fs laserpulses (120-fs, 800 nm, 80 MHz) using a pulse energyand beam dwell time ranging from 50 to 225 mW and10 to 250 ms, respectively. Contrary to the claim of100% transfection efficiency by Tirlapur and Konig,14

Stevenson measured an average transfection rate of50 ± 10% in 4000 laser-treated CHO cells.17 Thenonfluorescent dye, trypan blue, was used to confirmcell membrane viability.

In our lab, fs laser pulses were used to disrupt thecell plasma membrane for the purpose of introducingforeign substances into the cytoplasm of live MDCKcells. Kohli et al.2 showed that when fs laser pulseswere localized to the cell membrane, transient porescould be formed, exposing the extracellular spaceto the intracellular environment. Using a pulseenergy of 3 nJ/pulse (sub-10 fs, 800 nm, 80 MHz)cryoprotective disaccharides were cytoplasmically

Early fast phase (EFP)

0

(a) (b)

40 80Distance from targeted region (µm)

120

ControlPhoto-ablared

160

0.8

0.4

1.2

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0 40 80Distance from targeted region (µm)

120

ControlPhoto-ablared

160

0.8

0.4Rat

e of

CF

I (µm

/min

)

Rat

e of

CF

I (µm

/min

) 1.2

FIGURE 9 | (a) CFI rate measured 1 min after ablation (during early fast phase (EFP), solid line) at different distances from the ablated region, andcomparison with control embryos (squares, N = 3). (b) Same measurement 15 min after photoablation (during fast phase (FP)). Scale bar: 20 µm.Laser parameters: pulse duration 130-fs; excitation wavelength 830 nm; oscillator repetition rate 76 MHz; pulse energy for embryo manipulation0.6–4 nJ/pulse; 0.9 NA water immersion microscope objective. (Reprinted, with permission, from Ref. 16. Copyright 2005 The National Academy ofScience of the USA).



(b)

(d)

(f)

Stage 5

Stage 5

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Stage 7

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Stage 7

(a)

Stage 7

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(e)

(c)

FIGURE 10 | Multiphoton ablation allows quantified modulation ofspecific morphogenetic movements (a) and (b), control; (c) and (d),middorsal ablation, (e) and (f), postdorsal ablation). (a) Development ofan intact sGMCA embryo. Green represents images recorded at theequator. Red represents images recorded ≈ 20 µm under the surface.(c) Development of a sGMCA embryo after a 100 × 40 µm middorsalablation, resulting in disrupted lateral cell movements and no cephalicfurrow formation (gray arrowheads). (e) Development of a sGMCAembryo after 100 × 40 µm postdorsal ablation resulting in disruptedlateral cell movements only. (b),(d), and (f) Corresponding velocimetricanalysis for the same embryos at stage 7. Each experiment wasreproduced on five different embryos and gave similar results. Scale bar:100 µm. Black scale arrow, 5 µm/min. Laser parameters: pulse duration130-fs; excitation wavelength 830 nm; oscillator repetition rate 76 MHz;pulse energy for embryo manipulation 0.6–4 nJ/pulse; 0.9 NA waterimmersion microscope objective. (Reprinted, with permission, fromRef. 16. Copyright 2005 The National Academy of Sciences of the USA).

introduced through laser-induced transient pores forbiopreservation applications.2 When MDCK cells

were suspended in 1.0 m cryoprotective sucrose,the cells were found to swell to a new equilibriumvolume following transient pore formation as aresult of an intracellular accumulation of sucrose andwater (Figure 6(a, b)). The authors2 used volumetricanalyses to determine the longevity of the transientpore created in the cell membrane. Figure 6(c) depictsthe kinetics of the cell following permeabilization.Since the volumetric change was found to plateauwithin 200 ms (Figure 6(c)) it was hypothesized thatthis time corresponded to the lifetime of the laser-induced transient pore.2 The transient lifetime ofthe pore in varying molar concentrations was alsodetermined by the authors. A survival analysiswas performed using a membrane integrity assayconsisting of ethidium bromide and Syto 13. Inaddition, transport equations were used to estimatethe delivered intracellular concentration as a functionof the extracellular osmolarity.

In a recent study by Yanik et al.11 the authorsused fs laser pulses to perform laser axotomy of D-motor neurons in L4 larval-stage C. elegans. Severingof the D-neurons induced muscle contractions pre-venting backward locomotion. Figure 7 depicts timelapse images of the laser axotomy, where individualneurons were cut at the mid-body position using 100laser pulses at a pulse energy of 40 nJ/pulse (200-fs,1 kHz)11 (Figure 7(b)). The authors observed that thesevered neurons retracted following axotomy (Figure7(c, d)). Analysis of neuron regeneration revealed that54% of the laser-treated neurons (52 axons in 11worms) re-grew within 12–24 h (Figure 7(e)). A testof the motor neuron function showed that backwardlocomotion resumed within 24 h, with a functionalityapproaching that of wild type C. elegans.11

Chung et al.12 used fs laser pulses to studythe role of AFD neurons in C. elegans. Using apulse energy of 3 nJ/pulse (100-fs, 800 nm, 1kHz), individual dendrites within a bundle of amphiddendrites were severed.12 Severing of the dendriteswas accomplished without visible damage to adjacentdendrites. Similar to the observations made by Yaniket al., the ablated dendrites were found to retractfollowing ablation, with a retraction distance of5µm.12 To determine whether the dendrites re-grewafter laser dissection, the authors severed fluorescentlylabeled PHA and PHB sensory neurons and monitoredneuron growth for 24 h. In over 50 C. elegans, noneof the sensory neurons repaired, indicating that thecuts were permanent.12

Nishimura et al.13 used fs laser pulses tophotodisrupt microvessels in the parenchyma ofrat brains using a range of average laser powersfrom 0.03 to 0.5 µJ/pulse (100-fs, 1 kHz). Figure 8



FIGURE 11 | Middorsal ablationmodulates morphogenetic movements at theanterior pole, which are correlated with twistexpression. (a)–(f) Sequence of developmentat the anterior pole of control andphotoablated sGMCA embryos, showing thedisrupted movements of SP cells aftermiddorsal ablation. Approximate time afterthe onset of gastrulation is indicated inminutes (inverted contrast images). Blackscale arrow: 2 µm/min. Laser parameters:pulse duration 130-fs; excitation wavelength830 nm; oscillator repetition rate 76 MHz;pulse energy for embryo manipulation 0.6–4nJ/pulse; 0.9 NA water immersion microscopeobjective. (Reprinted, with permission, fromRef. 16. Copyright 2005 The NationalAcademy of Sciences of the USA).

(a) 8 min (stage 6)

Control

20 min (stage 6) (d) 8 min (stage 6)

(b) (e)

(c) (f)

CompressionExpansion

Photo-ablated

20 min (stage 6)

2PE

F

sequ

ence

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ocim

etric

anal

ysis

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orm

atio

n an

alys

is

(b) (c)

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FIGURE 12 | (a) When sub-10 fs laser pulses were focused through the chorion, laser-induced transient pores were created at theblastomere–yolk interface or in individual blastomeres of zebrafish embryos. Transient pores were formed only at the focus, leaving the chorion layerundamaged. The pores were used to introduce foreign material into the embryonic cells. Three-dimensional movement of the laser focal spot allowedfor precise targeting of any location on or within the embryo. (b) An early 8-cell stage embryo was targeted for pore formation at the blastomere–yolkinterface (arrow). (c) A sub-micron (∼800 nm) transient pore was created at the interface dividing the blastomeres (B) and yolk (Y) (arrow). Thesub-micron pore is obscured by a laser-generated cavitation bubble. An energy of 3 nJ/pulse at a gated pulse train of 200–300 ms was used to formthe pore. (d) Depicts the developing embryo at 64/128-cell stage 45–60 min post-fs laser poration. Scale bar for (b),(d) and (c) represents 200 µm and5 µm, respectively. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillator repetition rate 80 MHz; pulse energy forembryo manipulation 3 nJ/pulse; beam dwell time 200–300 ms; 1.0 NA 60× water immersion microscope objective. (Reprinted, with permission,from Ref. 3. Copyright 2007 Wiley Periodicals, Inc.).

depicts three different vascular lesions that wereproduced using varying pulse energies and pulsedensities. These included laser induced hemorrhaging,extravasation and intravascular clot formation13

(Figure 8). At relatively high laser pulse energiesabove the threshold for extravasation of fluorescentlylabeled blood plasma (0.03 µJ), hemorrhage of theblood plasma and red blood cells from the targetedvessel was observed.13 Lowering the pulse energyresulted in more controlled vascular lesions. However,both extravasation of intact vessels with continuedblood flow and clot formation resulting in completevessel obstruction were observed.13 The authors also

measured the changes in adjacent and downstreamblood flow in the obstructed vessel following laser-induced clot formation.

Embryo NanosurgeryWhile the application of fs laser pulses has beenextensively used in the nanosurgery of simple cells,the study of complex multicompartmental biologicalsystems such as embryos remains a challenge. Theability to noninvasively manipulate the intracellularenvironment of individual embryonic cells has



(b)(a)

FIGURE 13 | Brightfield and fluorescence images (a),(b) of adechorionated embryo at 16-cell stage that was fs laser porated in theblastomere cells for introducing FITC. Direct poration of the cellsresulted in a stronger FITC signal than poration at the blastomere–yolkinterface. The concentration of FITC used was 0.02–0.03 mg/ml. Theembryo was porated using an energy of 0.5–0.6 nJ/pulse at a gatedpulse train of 200–500 ms. Scale bar represents 200 µm. Laserparameters: pulse duration sub-10 fs; excitation wavelength 800 nm;oscillator repetition rate 80 MHz; pulse energy for embryo manipulation0.5–0.6 nJ/pulse; beam dwell time 200–500 ms; 1.0 NA 60× waterimmersion microscope objective. (Reprinted, with permission, fromRef. 3. Copyright 2007 Wiley Periodicals, Inc.).

important implications for future developments inmedical and developmental biology.

In a recent study by Supatto et al.,16 theauthors used fs laser pulses to induce morphogeneticmovements in Drosophila embryos. The authorsdemonstrated that laser nanosurgery (130-fs, 830nm, 76 MHz) below the vitelline membrane couldbe achieved within the developing embryo withoutdisturbing cytoskeletal dynamics adjacent to theablated area.16 A series of dissection line cuts, 100 ×40 µm, were made 5–15 µm beneath the vitellinemembrane with varying pulse energies and pulsenumber densities (number of incident fs laser pulsesper area). The dissections were characterized byobserving endogenous fluorescence emission usingtwo-photon excited fluorescence.16 For pulse densitiesand pulse energies below 105 µm−2 and 4 nJ/pulse,respectively, no endogenous fluorescence emissionwas observed.16 However, with increasing pulsedensity, fluorescence emission was observed along thedissection cut with microexplosions in the perinuclearregion of the cytoplasm. With a pulse densityapproaching 106 µm−2, large cavitation bubbles inexcess of 5–6 µm in diameter were observed.16

Further work examined the in vivo modulationof cellularization front invagination (CFI) in embryosablated by fs laser pulses.16 Figure 9 depicts therate of CFI in control and laser ablated Drosophilaembryos.16 As shown in Figure 9(a), an increase inthe rate of CFI was observed for the early fastphase one min after laser ablation, relative to the

controls. Fifteen minutes after ablation, no differencein the CFI rate for the fast phase was observed,16

Figure 9(b). Despite the increase in CFI for the earlyfast phase, the authors reported that kymographanalysis showed that cellularization completed incells adjacent to the laser ablated area. In additionto monitoring changes in the cellularization rate,the in vivo morphogenetic movements in embryostargeted at dorsal ablation sites were quantified.16

Figure 10 depicts the morphogenetic movementsand velocimetric analysis of the ablated and controlembryos. In Figure 10(a, b), both cephalic furrowformation and lateral cell motions were clearlyobserved in control embryos (arrows). However,middorsal dissection, (Figure 10(c, d)), resulted inno cephalic furrow formation and the disruption oflateral cell movements.16 Ablation of the postdorsalregion was found to affect the lateral cell movementsonly, (Figure 10(e, f)), with furrow formationoccurring normally. The authors speculated that themechanism responsible for the modulation likely arosefrom the disruption of the motor region associatedwith the ablated area.16 Further investigationsexamined cell movement and twist expression afterlaser ablation.16 When embryos were targeted atthe middorsal site, the stomodeal primordium (SP)cell motions were affected16 (Figure 11). In controlembryos, expansion and compression of the SP cellswere readily observed (Figure 11(a–c)) however, thismovement was suppressed by middorsal ablationand the loss of furrow closure16 (Figure 11(d–f)).While ventral cells at the anterior pole in controlembryos were found to have forward movement, SPcells exhibited more backward directed motion inablated embryos (Figure 11(d–f)). At ablation sitesother than middorsal, no significant changes in thetwist expression were observed.16

Recently, in our lab Kohli et al.3 used fslaser pulses (sub-10 fs, 800 nm, 80 MHz) tointroduce exogenous material into early stage cellsof live developing embryos. The animal modelsystem chosen was the zebrafish (Danio rerio), anaquatic vertebrate organism that is genetically anddevelopmentally closer to humans than the commoninvertebrate Drosophila melanogaster.29,30 Presently,zebrafish are used in the study of genetics, drugmonitoring, human disease, cardiac function andblood disorders.31–37 Figure 12(a) depicts the methodused for targeting individual embryonic cells ofthe developing zebrafish.3 In both chorionated anddechorionated embryos, the authors focused fs laserpulses with a pulse energy ranging from 0.56 to 2.7nJ/pulse to a location near the blastomere-yolk (B–Y)interface for transient pore formation3 (Figure 12(a)).



FIGURE 14 | Fluorescence images 30 minpost-fs laser poration of developed (a) 32-cell,(d) 512/1K-cell, and (g) 128/256-cell stagechorionated embryos that were targetedbeyond the chorion for introducingperivitelline-FITC into the blastomeres. Thebrightfield embryos were laser porated at(b) 8-cell, (e) 128-cell, and (h) 32–64-cell stage.Uptake of perivitelline-FITC is evident asfluorescence in (c), (f), and (i), where individualblastomere cells are clearly visible. The arrowsin (c), (f), and (i) point to the location wheretransient pores were formed. Concentration ofFITC used was 0.02–0.03 mg/ml. All embryoswere fs laser porated using an energy of 3nJ/pulse at a gated pulse train of 200–300 ms.Embryos were dechorionated to eliminate theinterfering fluorescence signal originating fromthe perivitelline space. Scale bar represents200 µm. Laser parameters: pulse durationsub-10 fs; excitation wavelength 800 nm;oscillator repetition rate 80 MHz; pulse energyfor embryo manipulation 3 nJ/pulse; beamdwell time 200–300 ms; 1.0 NA 60× waterimmersion microscope objective. (Reprinted,with permission, from Ref. 3. Copyright 2007Wiley Periodicals, Inc.).

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The authors first addressed whether the applied laserpulses were deleterious to the development of theembryo. Fs nanosurgery was performed at the B–Yinterface (Figure 12(b, c) arrows), in early cleavageto early blastula (2-cell to 128-cell) stage embryosusing a pulse energy of 2.7 nJ/pulse and a beamdwell time of 200–500 ms. Figure 12(d) shows normaldevelopment of a laser treated 8-cell stage embryo,which has developed to 128-cell stage 45–60 min post-laser surgery.3 Other targeted embryos were found todevelop normally as compared to control embryos. Todetermine if laser surgery at the B–Y interface or onindividual blastomere cells lead to the formation of atransient pore, the authors suspended early cleavage toearly blastula (2-cell to 128-cell) stage dechorionatedembryos in a fluorescent reporter molecule, fluoresceinisothiocyanate (FITC), and examined fluorescenceuptake in the embryonic cells. Figure 13 depicts FITCfluorescence in the blastomere cells of a 16-cell stageembryo, confirming transient pore formation andexogenous material delivery. In 39 targeted embryos,a FITC loading efficiency of 87% was reported.3 Itwas conjectured that the distribution of the fluorescentprobe to adjacent blastomeres likely occurred throughblastomere bridges or gap junctions, depending on thedevelopmental stage.38–40

Figure 12(a) depicts the chorion, a proteinaceousmembrane surrounding the developing embryo, whichprovides protection from the environment. To showthat the applied laser pulses could still be focusedfor pore formation at the B–Y interface, the authorsfocused fs laser pulses beyond the structure of thechorion as shown in Figure 12(a). Early cleavage toearly blastula (2–cell to 128-cell) stage chorionatedembryos were suspended in the presence of FITC, andthe fluorescent probe was allowed diffuse into theperivitelline space (FITC was previously shown to beimpermeable to the blastomeres). Targeting the B–Yinterface with a pulse energy of 2.7 nJ/pulse and abeam dwell time of 200–300 ms, the authors foundthat they could introduce perivitelline FITC into theembryonic cells without compromising the structureof the chorion.3 This is evident in Figure 14(c, f,i), where after proteolytic digestion of the chorion(to remove the interfering fluorescent signal from theperivitelline region), fluorescence was observed in theindividual blastomere cells. In a total of 27 laser-treated embryos, a FITC loading efficiency of 78%was found.3

Exogenous material delivery was not limited toFITC, as the authors also demonstrated the delivery ofconjugated quantum dots and plasmid DNA. Quan-tum dots and DNA are important materials that have



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FIGURE 15 | (a) An early 2-cell stage dechorionated embryo thatwas fs laser porated in the blastomere cells for introducingStreptavidin-conjugated quantum dots. The quantum dots freelydiffused throughout the cells and remained fluorescent as the embryodeveloped. (b) Depicts the same embryo developed past germ ring.Concentration of the Streptavidin-conjugated quantum dot solution was0.3 µM. An energy of 1.5–2 nJ/pulse at a gated pulse train of 200–500ms was used, and 3–4 pores were created in each cell for introducingthe quantum dots. Fluorescence and brightfield images of 24 hpf larvae,(c)–(f), expressing the sCMV-EGFP construct that was introduceddirectly into the blastomere cells of an early to mid cleavage stage(2-cell to 8/16-cell) dechorionated embryo. (c),(d) Expression isobserved along the gut, as well as in the floor plate, and somites.(e),(f) Expression of sCMV-EGFP is seen throughout the tail of the larva,where expressing cells are those near the floor plate and somites.Concentration of the construct used was 170 µg/ml. An energy of0.5–0.6 nJ/pulse at a gated pulse train of 200–500 ms was used, with3–4 pores created per cell for introducing the plasmid (maximum of 2,2, 4, and 8 cells targeted per 2–, 4–, 8–, and 16-cell stage respectively).Laser parameters: pulse duration sub-10 fs; excitation wavelength 800nm; oscillator repetition rate 80 MHz; pulse energy for embryomanipulation 0.5–2 nJ/pulse; beam dwell time 200–500 ms; 1.0 NA60× water immersion microscope objective. (Reprinted, withpermission, from Ref. 3. Copyright 2007 Wiley Periodicals, Inc.).

potential uses for cell fate mapping and the develop-ment of stable transgenic fish lines.41,42 Using a pulse

energy of 1.5–2 nJ/pulse, streptavidin-conjugatedquantum dots (targeted near the B–Y inter-face) were introduced into 2-cell stage dechorionatedembryos3 (Figure 15(a, b)). Quantum dot fluorescencewas observed in the early embryonic cells (Figure 15a)while in later cell stages up to germ ring (Figure 15b)the quantum dots were visibly dispersed throughoutthe blastomeres.3 To determine if the applied laserpulses constituted a valid alternative method forDNA delivery, the authors laser transfected early tomid cleavage (2-cell to 8/16-cell) stage dechorionatedembryos in the presence of a circular plasmid, sCMV-EGFP, with a pulse energy of 0.56 nJ/pulse and a beamdwell time ranging from 200 to 500 ms.3 Expressionof the plasmid construct was observed in a 24-hpost-fertilization (hpf) larva (Figure 15(c, e)) withthe expression seen along the yolk-extension, floorplate, somites and tail cells of the larva.3 In over 45chorionated and dechorionated laser treated embryos,survival approached 90%, with embryo morphologyand behavior similar to the control sample.3

Kohli and Elezzabi15 further examined thedevelopment of zebrafish embryos after laser surgerywith the fs laser (sub-10 fs, 800 nm, 80 MHz). Usinga pulse energy of 0.56 nJ/pulse and a beam dwelltime of 100 ms, individual chorionated blastomerecells were surgically ablated at the early 2-cell stagein over 40 embryos.15 Each blastomere cell wasablated at three different locations with a total laserexposure time of 300 ms per targeted site. The authorsreared the embryos to 2 and 7 days post-fertilization(dpf) and used light microscopy (LM) and scanningelectron microscopy (SEM) to determine if the appliedlaser pulses induced morphological changes in thedevelopment of the embryos. Under LM, the bodyplans of control and laser-manipulated embryos wereinspected with emphasis on the development of thebody axis.15 Short-term survival (before 2 dpf), asdetermined by the above analysis, revealed a survivalpercentage of 93%.15 Viable larvae showed no differ-ences in developmental or hatching rates as comparedto the controls. SEM imaging showed key develop-mental structures including the caudal fin, dorsal fin,yolk sac extension, yolk sac and the olfactory pit tobe morphologically similar in laser-manipulated andcontrol larvae.15 As the laser-treated larvae aged, thepectoral fin buds lifted away from the yolk sac anddeveloped into mature pectoral fins along the lateralextent of the zebrafish body. This morphologicaldevelopment was consistent with control larvae.15

The authors concluded that no short-term effects ofthe laser on the development were observed.

While no short-term effects were observed,Kohli commented that the laser’s effect on embryonic



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FIGURE 16 | Key developmental structures in a laser-manipulated and a control larva reared to 7 dpf. (a) Inverted whole body image of alaser-manipulated larva at 7 dpf. Structures indicated are the ventral fin (VF), notochord (NC), pectoral fin (PF), otic capsule (OC), otic vesicle (OV),eye (E), olfactory pit (OP), and the protruding mouth (PM). (b) Inverted whole body image of a control larva at 7 dpf. Similar developmental structuresobserved in (a) were also seen in (b). (c) Kinocilia projecting from the lateral crista of a laser-manipulated and (d) control larva. Scale bar for(a),(b) represent 200 µm and (c),(d) 1 µm, respectively. Laser parameters: pulse duration sub-10 fs; excitation wavelength 800 nm; oscillatorrepetition rate 80 MHz; pulse energy for embryo surgery 0.56 nJ/pulse; total beam dwell for laser-manipulation 300 ms; 1.0 NA 60× water immersionmicroscope objective.

development may not become apparent until laterdevelopmental stages.15 Using SEM, the authorsexamined control and laser-manipulated larvae rearedto 7 dpf. Developmental structures inspected includedthe protruding mouth, olfactory pit, pectoral fin,eye, otic capsule, otic vesicle, ventral fin, notochord,posterior forebrain, and dorsal midbrain.15 Nodifferences in the placement or patterning of thesestructures were observed between the samples. Figure16(a, b) depict mosaics of a laser-manipulated anda control larva with the developmental structuresindicated as mentioned above. High magnificationimages revealed that the olfactory pit in the controland laser-manipulated larvae was surrounded byepidermal cells, with the pit rims covered by longkinocilia.15 In the lumen of the eara crista was foundon the lateral wall with kinocilia projecting from thesensory epithelial, as seen in Figure 16 (c, d).15 Theauthors found no differences in neuromast patterning,with projecting kinocilia that were distributed alongthe lateral line of the zebrafish body.15 In controllarvae, neuromasts were found anterior to theolfactory pit, at the outer rim of the otic capsule,anterior to the diencephalon and adjacent to bothsides of the optic tectum (dorsal midbrain) anddiencephalons (posterior anterior-forebrain).15 Theneuromast patterning in laser-manipulated larvae wasfound to be identical to that seen in the controls. It wasconcluded that no long-term developmental effectscould be observed, thereby making the application offs laser pulses an important noninvasive tool for thestudy of live embryos. Further work is being conducted

by the authors to determine if any physiologicalresponses are induced following fs laser nanosurgery.

CONCLUSIONThe noninvasive nature of fs laser pulses and theirability to target subcellular sites with high spatialresolution are the major features that have made theseultrafast lasers an attractive tool for the study of livecells and embryos. This review article has exploreddevelopments in cell and embryo nanosurgery; eachreported study identified unique applications tobiology. These include the knockdown of subcellularorganelles, the opto-injection of exogenous materials,and functional analyses of laser-induced morpho-genetic and morphological changes in embryonicdevelopment. However, despite these advances, thefull potential of fs laser pulses has yet to be realized.Like the laser itself, the fs laser is a tool being devel-oped without having fully elucidated all of its potentialuses. In addition, the application of fs laser pulses isin some ways developing as a technique ‘in search of abiological problem’. It is through continued researchthat we will uncover novel applications that willundoubtedly benefit many biological disciplines. Weenvision that in the near future fs laser pulses will beused in the study of cell fate mapping to identify howindividual cells contribute to the overall embryonicdevelopment of organisms. It will be possible to cry-opreserve embryos with low solute permeabilities bydelivering impermeable and permeable cryoprotectiveagents. The generation of genetically modified



organisms will be possible as a result of the inter-ference of delivered exogenous nucleic acids, as willbe the potential development of stable transgenic celllines. The collaboration of physicists, engineers, and

cell and developmental biologists will enable the pur-suit of such applications, with the fs laser providing anew prospective for understanding essential biologicalsystems.

REFERENCES

1. Kohli V, Acker JP, Elezzabi AY. Cell nanosurgery usingultrashort (femtosecond) laser pulses: applications tomembrane surgery and cell isolation. Lasers Surg Med2005, 37:227–230.

2. Kohli V, Acker JP, Elezzabi AY. Reversible permeabi-lization using high-intensity femtosecond laser pulses:applications to biopreservation. Biotechnol Bioeng2005, 92(7):889–899.

3. Kohli V, Robles V, Cancela ML, Acker JP, Wask-iewicz AJ, et al. An alternative method for deliveringexogenous material into developing zebrafish embryos.Biotechnol Bioeng 2007, 98(6):1230–1241.

4. Konig K, Riemann I, Fritzsche W. Nanodissection ofhuman chromosomes with near-infrared femtosecondlaser pulses. Opt Lett 2001, 26(11):819–821.

5. Tirlapur UK, Konig K. Femtosecond near-infrared laserpulses as a versatile non-invasive tool for intra-tissuenanoprocessing in plants without compromising viabil-ity. Plant J 2002, 31(3):365–374.

6. Shen N, Datta D, Schaffer CB, LeDuc P, Ingber DE,et al. Ablation of cytoskeletal filaments and mitochon-dria in live cells using femtosecond laser nanoscissor.MCB 2005, 2(1):17–25.

7. Watanabe W, Arakawa N, Matsunaga S, Higashi T,Fukui K, et al. Femtosecond laser disruption of sub-cellular organelles in a living cell. Opt Express 2004,12:4203–4213.

8. Sacconi L, Tolic-Norrelykke IM, Antolini R,Pavone FS. Combined intracellular three-dimensionalimaging and selective nanosurgery by a non-linear microscope. J Biomed Opt 2005,10(1):):014002-1–014002-5.

9. Heisterkamp A, Maxwell IZ, Mazur E, Under-wood JM, Nickerson JA, et al. Pulse energy dependenceof subcellular dissection by femtosecond laser pulses.Opt Express 2005, 13(10):3690–3696.

10. Konig K, Riemann I, Fischer P, Halbhuber K-J. Intra-cellular nanosurgery with near infrared femtosecondlaser pulses. Cell Mol Biol 1999, 45(2):195–201.

11. Yanik MF, Cinar H, Cinar HN, Chisholm AD, Jin Y,et al. Functional regeneration after laser axotomy.Nature 2004, 432:822.

12. Chung SH, Clark DA, Gabel CV, Mazur E,Samuel AD. The role of the AFD neuron in C. ele-gans thermotaxis analyzed using femtosecond laserablation. BMC Neurosci 2006, 7:30.

13. Nishimura N, Schaffer CB, Friedman B, Tsai PS,Lyden PD, et al. Targeted insult to subsurface corticalblood vessel using ultrashort laser pulses: three modelsof stroke. Nat Methods 2006, 3(2):99–108.

14. Tirlapur UK, Konig K. Targeted transfection by fem-tosecond laser. Nature 2002, 418:290.

15. Kohli V, Elezzabi AY. Laser surgery of zebrafish (Daniorerio) embryos using femtosecond laser pulses: optimalparameters for exogenous material delivery, and thelaser’s effect on short- and long-term development.BMC Biotechnol 2008, 8(7):1–20.

16. Supatto W, Debarre D, Moulia B, Brouzes E, MartinJ-L, et al. In vivo modulation of morphogenetic move-ments in Drosophila embryos with femtosecond laserpulses. PNAS 2005, 102(4):1047–1052.

17. Stevenson D, Agate B, Tsampoula X, Fischer P,Brown CTA, et al. Femtosecond optical transfectionof cells: viability and efficiency. Opt Express 2006,14(16):7125–7133.

18. Oraevsky AA, Silva LBD, Rubenchik AM, Feit MD,Glinsky ME, et al. Plasma mediated ablation of bio-logical tissues with nanosecond-to-femtosecond laserpulses: relative role of linear and nonlinear absorption.IEEE J Quantum Elect 1996, 2(4):801–809.

19. Vogel A, Noack J, Huttman G, Paltauf G. Mechanismsof femtosecond laser nanosurgery of cells and tissues.Appl Phys B 2005, 81:1015–1047.

20. Niemz M. Laser-tissue Interactions: Fundamentals andApplications. Springer, ed. Berlin, Heidelberg, NewYork: Springer-Verlag; 2002.

21. Loesel FH, Fischer JP, Gotz MH, Horvath C, Juhasz T,et al. Non-thermal ablation of neural tissue with fem-tosecond laser pulses. Appl Phys B 1998, 66:121–128.

22. Noack J, Vogel A. Laser-induced plasma formationin water at nanosecond to femtosecond time scales:calculation of thresholds, absorption coefficients,and energy density. IEEE J Quantum Elect 1999,33(8):1156–1167.



23. Schaffer CB, Brodeur A, Mazur E. Laser-inducedbreakdown and damage in bulk transparent materi-als induced by tightly focused femtosecond laser pulses.Meas Sci Technol 2001, 12:1784–1794.

24. Kaiser A, Rethfeld B, Vicanek M, Simon G. Micro-scopic processes in dielectrics under irradiation bysubpicosecond laser pulses. Phys Rev B 2000,61(17):11437–11450.

25. Sacchi CA. Laser-induced electric breakdown in water.J Opt Soc Am B 1990, 8(2):337–345.

26. Boyle JW, Ghormley JA, Hochanadel CJ, Riley JF. Pro-duction of hydrated electrons by flash photolysis ofliquid water. J Phys Chem 1969, 73(9):2886–2890.

27. Arnold CL, Heisterkamp A, Ertmer W, LubatschowskiH. Computational model for nonlinear plasma for-mation in high NA micromachining of transpar-ent materials and biological cells. Opt Lett 2007,15(16):10303–10317.

28. Venugopalan V, Guerra A III, Nahen K, Vogel A. Roleof laser-induced plasma formation in pulsed cellularmicrosurgery and micromanipulation. Phys Rev Lett2002, 88(7):078103-1–078103-4.

29. Vogel G. Zebrafish earns its stripes in genetic screens.Science 2000, 288(5469):1160–1161.

30. van der Sar AM, Appelmelk BJ, Vandenbroucke-Grauls CMJE, Bitter W. A star with stripes: Zebrafishas an infection model. Trends Microbiol 2004,12(10):451–457.

31. Barut BA, Zon LI. Realizing the potential of zebrafishas a model for human disease. Physiol Genomics 2000,2:49–51.

32. Warren KS, Wu JC, Pinet F, Fishman MC. The geneticbasis of cardiac function: Dissection by zebrafish(Danio rerio) screens. Philos Trans R Soc Lond B2000, 355:939–944.

33. Dooley K, Zon LI. Zebrafish: A model system for thestudy of human disease. Curr Opin Genet Dev 2000,10:252–256.

34. Hill AJ, Teraoka H, Heideman W, Paterson RE.Review: zebrafish as a model vetebrate for investigatingchemical toxicity. Toxicol Sci 2005, 86(1):6–19.

35. Jagadeeswaran P, Sheehan JP. Analysis of blood coag-ulation in the zebrafish. Blood Cells Mol Dis 1999,25(15):239–249.

36. Nasevicius A, Ekker SC. Effective targeted gene ‘knock-down’ in zebrafish. Nat Genet 2000, 26:216–220.

37. Thisse C, Zon LI. Organogenesis-heart and blood for-mation from the zebrafish point of view. Science 2002,295:457–462.

38. Kimmel CB, Law RD. Cell lineage of zebrafish blas-tomeres, I. Cleavage pattern and cytoplasmic bridgesbetween cells. Dev Biol 1985, 108(1):78–85.

39. Kimmel CB, Ballard WW, Kimmel SR, Ulmann B,Schilling TF. Stages of embryonic development of thezebrafish. Dev Dyn 1995, 203:253–310.

40. Weinberg ES. Analysis of early development in thezebrafish embryo. In: Hennig W. ed., Early EmbryonicDevelopment of Animals. Berlin, Heidelberg: Springer-Verlag; 1992, 91–150.

41. Rieger S, Kulkarni RP, Darcy D, Fraser SE, Koster RW.Quantum dots are powerful multipurpose vital label-ing agents in zebrafish embryos. Dev Dyn 2005,234:670–681.

42. Higashijima S-I, Okamoto H, Ueno N, Hotta Y,Eguchi G. High-frequency generation of transgeniczebrafish which reliably express GFP in whole mus-cles or the whole body by using promoters of zebrafishorigin. Dev Biol 1997, 192:289–299.

FURTHER READING

Zipfel WR, Williams RM, Webb WW. Nonlinear magic: Multiphoton microscopy in the biosciences. NatBiotechnol 2003 21(11) 1369–1377.Debarre D, Supatto W, Pena A-M, Fabre A, Tordjmann T, et al. Imaging lipid bodies in cells and tissues usingthird-harmonic generation microscopy. Nat Methods 2006 3(1) 47–53.Watanabe W, Shimada T, Matsunaga S, Kurihara D, Fukui K, et al. Single-organelle tracking by two-photonconversion. Opt Express 2007 15(5) 2490–2498.

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