Julien Colombelli, Stephan W. Grill and Ernst H. K. Stelzer- Ultraviolet diffraction limited...

$: Julien Colombelli, Stephan W. Grill and Ernst H. K. Stelzer- Ultraviolet diffraction limited nanosurgery of live biological tissues$
Ultraviolet diffraction limited nanosurgery of live biological tissuesJulien Colombellia)

European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

Stephan W. GrillMPI-CBG, Pfotenhauerstrasse 108, D-01307 Dresden, Germany

Ernst H. K. StelzerEuropean Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany

~Received 25 September 2003; accepted 15 October 2003!

A laser nanodissection system forin vivo and in situ biological tissues is presented. A pulsed laserbeam operating at a wavelength of 355 nm enables diffraction limited dissection, providing anoptimal tool for intracellular nanosurgery. Coupled into a conventional inverted microscope andscanned across a field of up to 1003100mm2, this optical nanoscalpel performsin vivophotoablation and plasma-induced ablation inside organisms ranging from intracellular organelles toembryos. The system allows the use of conventional microscopy contrasts and methods, fastdissection with up to 1000 shots per second, and simultaneous dissection and imaging. This articleoutlines an efficient implementation with a small number of components and reports animprovement of this state of the art of plasma-induced ablation technique over previous studies,with a ratio of plasma volume to beam focal volume of 5.2. This offers, e.g., the possibility ofwriting information directly at the sample location by plasma glass nanopatterning. ©2004American Institute of Physics.@DOI: 10.1063/1.1641163#

I. INTRODUCTION

Laser microsurgery of biological tissues1–3 has beenstudied for over 30 years but is still a field of thorough re-search and major controversies. The mechanisms of laser-tissue interactions have been extensively investigated.3–10

The underlying physical principles arephotoablationandplasma-induced ablation. Photoablation4–6 occurs by meansof photochemical reactions. The energy of UV photons issufficiently high~typically .3.5 eV! to reach the moleculardissociation energy and, therefore, to break chemical bonds.This leads to the photodecomposition of the irradiated mol-ecules. For biological applications, the most suitable wave-lengths in this respect are in the UV-A range~315 nm,l,400 nm!, since UV-B ~280 nm,l,315 nm! is stronglyabsorbed by DNA and provokes irreversible damage and mu-tations in living samples. UV-C~100 nm,l,280 nm! ab-sorption is too high in turbid media and would also requirespecial optical elements, thus raising the cost and decreasingthe flexibility of an irradiation setup based on a conventionalmicroscope.

Plasma formation occurs above a certain energy orpower density threshold3,5,11,12 when ionization of the me-dium starts by means of thermal or multiphoton ionization.Ablation occurs inside the plasma volume, the shape and sizeof which depend on the beam geometry. The energy densitythreshold for ionization has been found to depend on thepulse duration.11,12 Below the ns range, the threshold in en-ergy density decreases and microplasmas can be inducedwith significantly less energy than with the nanosecond

pulses commonly utilized in commercial microdissectionsystems.13 This results in delicate ablation, i.e., mechanicalside effects like shockwave or cavitation, likely to damagethe tissue structure far outside the ablating spot area, can bereduced or avoided.

With this in mind, a nanodissection system was imple-mented on a conventional inverted microscope. Using apulsed UV laser with sub-ns pulse width and a high numeri-cal aperture~NA! lens, low energy ablation~due to the shortpulse! in a highly confined volume~due to the short wave-length of 355 nm! is possible. Peak power densities of up to10 TW cm22 are achieved with a low energy laser source~8.8 mJ per pulse!. Potential mechanical or thermal damageto the living samples outside the focal volume are avoidedduring this ablation process. The setup combines featuresfrom available commercial systems and overcomes many oftheir limitations. In particular, we achieve diffraction limitedfocusing with short sub-ns ablation~avoiding severe thermaland mechanical damages to living samples!, fast beam scan-ning, and optimal coupling. This allows simultaneousin vivodissection and image acquisition. All microscopy modes re-main available, including fluorescence and a port for a con-focal module. The working distances of available microscopeobjective lenses allows one to perform subcellular surgery aswell as thick sample dissection~e.g., in living embryos!.Here, we present a detailed description of this device, discussthe technical accuracy, speed, and potential biological appli-cations, and present the option to store information directlyat the sample site by means of plasma-induced glass pattern-ing inside the sample holder~e.g., cover slip!.a!Electronic mail: [email protected]

REVIEW OF SCIENTIFIC INSTRUMENTS VOLUME 75, NUMBER 2 FEBRUARY 2004

4720034-6748/2004/75(2)/472/7/$22.00 © 2004 American Institute of Physics

Downloaded 03 Feb 2004 to 194.94.44.4. Redistribution subject to AIP license or copyright, see http://rsi.aip.org/rsi/copyright.jsp

http://dx.doi.org/10.1063/1.1641163

II. INSTRUMENT

A. Overview

The instrument is based on a Zeiss inverted microscopeAxiovert 200M as shown in Fig. 1. No major hardwaremodification is necessary to achieve a high performancesetup. The main parts are listed in Table I together with theirspecifications. In the setup, a pulsed UV laser Nd:YAG atl5355 nm~third harmonic oflD51064 nm) is expanded bymeans of telecentric optics to meet the diffraction limit witha Zeiss 633/1.2W lens. The theoretical beam diameter in thefocal plane is 361 nm. A galvanometer pair scan unit guidesthe laser beam across a field of 1003100mm2. Coupling

into the microscope is achieved via the fluorescence illumi-nation path. The field aperture slider has been modified andcontains a dichroic mirror reflecting at the laser wavelengthof 355 nm. The laser power is controlled by an acousto-optical tunable filter~AOTF!. Because most objective lensesare not designed for UV-A illumination, chromatic aberra-tions are expected and will cause the UV laser focus to beshifted axially relative to the visible light focus. These focalshifts can be corrected by up to 100mm using a piezoelectricpositioner. Both the AOTF and the piezoelectric positionerare driven via the computer’s RS232 interface communicat-ing with a Scanning Controller DSP board triggering the la-

FIG. 1. Two schematic representa-tions ~a! and ~b! and a photograph~c!of the UV nanoirradiation setup, thesecond scheme~b! shows the opticalpath from above. The power of the fre-quency tripled Nd:YAG laser~1! iscontrolled by an AOTF~2!. The beamis expanded by a telecentric UV lens~3, magnification33!, hits a galvo-mirror pair ~4! and is coupled into anAxiovert 200M microscope~10! withtwo telecentric UV singlet lenses~5,resulting magnification factor32!and a dichroic mirror mounted on aslider ~7! at the field aperture location.Arrows indicate the orientation of thelaser light’s polarization. The systemtransmission is optimized using ans-polarized beam hitting the fluores-cence beam splitter~8!. Simultaneousdissection and fluorescence image ac-quisition is possible by placing theexcitation filters at the back of thefluorescence port in a filter wheel~6!.Other elements:~9! objective1piezo-electric positioner,~11! DSP board andthe galvanometer’s analog drivers,~12! fluorescence lamp1shutter.

TABLE I. Main components used in the nanodissection setup with their corresponding manufacturer and mainspecifications.

PartManufacturerpart number Specification

Pulsed Nd:YAG laser JDS Uniphase PowerChip PNV-001025-050

l5355 nm, energy per pulse 10mJ,pulse width 500 ps, repetition rate 1 kHz

UV Acousto-opticaltunable filter~AOTF!

AA optoelectroniqueAOTF.4C-UV

Transmission at 355 nm: 92%

UV beam expander Sill Optics S6ASS3103/075 Magnification33Galvanometer mirrors GSI Lumonics XY10 Optical scan angle6175 mrad, position

accuracy 50mrad,a repeatability 10mrada

Piezoelectric objective positioner Physik Instrumente~PIFOC! Positioning range 100mm,traveling time,1 ms

aValues specified by the manufacturer.

473Rev. Sci. Instrum., Vol. 75, No. 2, February 2004 UV diffraction limited nanosurgery


ser pulses and synchronizing them with the galvo-mirrorsteps. Finally, we provide a user interface by the means of agraphical software application embedded in the CCCsoftware.14 To control the irradiation sequence, the user maycontrol the following parameters: pulse energy~up to 8.8mJ!, number of pulses, repetition rate~up to 1 kHz!, andfocusz-position correction~up to 100mm!. A custom targetshape is defined on a live window across the full irradiationfield, and the scan controller board converts the graphicalcoordinates to angular coordinates that position the laserbeam pulses inside the sample. The setup offers all com-monly used microscopy contrast modes~fluorescence, differ-ential interference contrast or DIC, phase contrast as well asconfocal microscopy! in vivo and also permits the use ofdifferent objective lenses.

B. Coupling and scanning the UV beam

The coupling of UV-A light into a conventional micro-scope via the fluorescence path does not interfere with theavailable optical contrasts. In order to allow simultaneousdissection and imaging, the field aperture slider of the micro-scope contains a dichroic mirror with a high reflectance at355 nm and high transmittance in the visible spectrum. Forstandard epifluorescence the classical excitation lines in thevisible spectrum must be efficiently transmitted: in our case,70% transmission at 400 nm~Optosigma, USA! and morethan 95% from 450 to 700 nm. Excitation filters are removedfrom the filter block and inserted into a filter wheel betweenthe arc lamp and the microscope chassis to allow the propa-gation of UV laser light from the field aperture to the objec-tive lens. Using a Zeiss C-Apo 1.2W lens, diffraction limitedfocussing is achieved with a 3-times beam expander andscanning lenses~magnification32!, leading to a scan field of80380mm2 ~with an optical scan angle of 7.5°'131 mradat the galvanometric mirrors!. The accuracy and repeatabilityof the scanning mirrors depend mainly on the electronics. Weachieve a positioning accuracy of 10mrad ~corresponding to6 nm in sample plane! with 16-bit digital control and stableanalog drivers. The thermal noise of the electronics results ina steering accuracy of 50mrad ~30 nm!. Thus the positioningaccuracy is significantly better than the beam diameter in thefocal plane~by an expected factor of 60!.

In a previous version of this setup,15,16 the microscopestage was controlled to move the sample relative to thebeam. This solution has the advantage that the beam remainssteady at the center position of the field and is therefore lesslikely to suffer from spherical aberrations. Moreover, withthis type of sample scanning, irradiation can also be per-formed across a larger field of view.

In our setup, we achieve diffraction limited focusing us-ing a Zeiss C-Apo 633/1.2W water immersion lens. Asimple method for measuring the beam dimensions in theobject plane is illustrated in Fig. 2. The UV beam was fo-cused inside a conventional glass coverslip and the visibleeffect of one emitted pulse is observed in brightfield trans-mission mode for two different optical powers. The mini-mum detectable effect occurred with a pulse energy of 0.460.1mJ deposited in the medium, or 65 GW cm22 of peak

power density threshold. Figures 2~a! and 2~b! show a spotwith a diameterdxy50.4560.05mm in theXY plane and alength of l z52.5060.25mm alongZ. Figure 2~c! shows theeffect of a pulse with the maximum available energy, 8.860.1 mJ: a glass fracture extruding over several microns isproduced. To characterize the beam focus quality, the mea-surements at 0.4mJ can be compared to theoretical values.To estimate the dimensions of the focal volume, we considerit to be an ellipsoid with lateral extentDxy and elongationLz . The formulas appropriate for high NA lenses:17

Dxy52l/n~12cosa! ~1!

and

Lz52l/n~322 cosa2cos 2a!1/2, ~2!

wherea is the angle used in the definition of the numericalaperture NA5n sina, and n is the refractive index of thesample medium. The resulting volumeVf5p Dxy

2 Lz/6 of thefocal ellipsoid isVf50.050mm3, and can be compared tothe affected measured volume in glassVg50.265mm3. Wetherefore characterize the beam quality with the factorVg /Vf55.2.

Table II lists the accuracy and working conditions of thesystem with different Zeiss objective lenses that have a trans-mission of at least 50% at 355 nm, in order to minimize theUV losses. The differences in focal extent are directly due todifferences in numerical aperture of the focused beams. Us-ing a NA50.6 air lens instead of a NA51.2 water immersionlens, the size of the focus is increased by a factor of 2, butthe working distance is improved by seven times.

Finally, in order to improve the coupling of the 355 nmlaser line into the microscope and to optimize the systemtransmission, the beam polarization must be considered.Conventional filter sets are not designed to fit a special re-quirement at 355 nm so their polarization dependence can bequite complicated. In general, the beam splitter’s reflectivityis higher with an incidents-polarized beam on the beamsplitter. In Fig. 1, the arrows represent the orientation of theUV beam’s polarization, and the coupling and scanning op-tical elements are arranged to achieve ans-polarized beam.Nevertheless, standard commercial filters will not always re-flect sufficient power to perform nanosurgery. Two solutionscan be used to achieve high reflectance. First, the beam split-ter can be coated with a special layer that reflects the 355 nm

FIG. 2. Visible effect of pulsed UV laser-glass interaction. Above a certainpeak power density threshold~a! and~b! of ca. 65 GW cm22, glass proper-ties change inside the focal volume of the focused beam and the irradiationvolume becomes visible. At higher powers in~c!, mechanical side effectsinduced by photodisruption provoke glass fracture. Images were taken withbright-field transmission, therefore the dark and light zones do not providedirect information about the material property but only represent the reflec-tion of light by the modified glass material.~b! The image was extractedfrom a stack ofX/Y images recorded along the optical axis. Scale bar 5mm.

474 Rev. Sci. Instrum., Vol. 75, No. 2, February 2004 Colombelli, Grill, and Stelzer


line. Second, one can use double-band filter sets specifiedfor two chromophores, one in the near UV such as 48,6-Diamidino-2-phenylindole~or DAPI!, to ensure high reflec-tance of the beam splitter, and the other for the chromophoreof interest. In our setup, a maximum laser power in the focuscan reach up to 40% of the total output laser power, since weuse a beam splitter optimized for the 355 nm line and anobjective lens with high transmission. This means that up to8.8 mJ can be deposited in the sample per pulse, correspond-ing to an estimated peak power density of about 10 TW cm22

with a diffraction limited beam.

C. Synchronization and software

The software interface of the system is based on thesoftware for the Compact Confocal Camera.14 Users outlinea target on a live image and control laser output power, num-ber of laser pulses, objective defocus, and mirror pair scan-ning speeds. The dynamic behavior of the system is limitedby the optimal laser repetition rate of 1 kHz. Any point in thescan field can thus be reached and irradiated once every mil-lisecond. The digital controller for the mirrors, the AOTF,and the piezoelectric positioner are controlled via the RS232interface of a PC. The standard features of the microscopeoperate independently of the irradiation protocol, i.e., itshardware does not have to be controlled.

In Fig. 3, a block diagram shows the logical sequence ofa typical irradiation procedure together with the hardware

connections. Figure 4 presents the timing structure of thissequence. After the user has set the parameters for dissection,target, and the acquisition~e.g., how many images beforeand after the irradiation are captured!, the nanosurgery pro-cess is started. The irradiated positions are downloaded to theScan Controller board SC2000 at a speed of 115.2 kB/s andare converted to angle values for the scan mirrors. The du-ration of this download was measured to lastDt15(6.1n148) ms, wheren is the number of shots. The piezoelectricobjective positioner then places the UV beam focus in theimage plane~i.e., the chromatic shift of the UV focus iscompensated!. The surgical sequence is executed by theSC2000, which synchronizes the laser pulses with the mir-rors steps at a frequencyf up to 1 kHz, and therefore lasts atime period ofDt15n/ f . The objective positioner is thenmoved back so that the image plane is in focus. The totaltime interval between the irradiation command and the endof the dissection procedure isDt5(6.1n1n/ f 163) ms. Forexample, the whole protocol for irradiating a hundred pulsesalong a line at 1 kHz would last 773 ms, with only 10 msbeing used for the optical dissection sequence. A single pulseirradiation is achieved in about 70 ms. The acquisition runsin parallel with an IEEE 1394 charge coupled device camera,triggering a shutter for fluorescence mode as shown in Fig. 3.One alternative to this beam-scanning system is to directlydrive the stage of the microscope in order to move thesample relative to a fixed beam. This solution always offers

TABLE II. Different objective lenses used for 355 nm nanodissection system. Diffraction limited focusing is achieved under particular conditions with highNA lenses. Using a lower NA, the beam spot accuracy is decreased by a factor of 2, but the working distance can be improved by seven times for thicksamples’ applications.

Objective lens~Zeiss!Theoretical

Airy disk ~nm!MeasuredX-Y spot

accuracy~nm!Free workingdistance~mm!

Focus aberrationat 355 nm~mm!

Specifiedtransmission~%!

Measuredscanning field~mm!

C-Apo 633/1.2 W 361 ,450 0.24 4.060.5 .50 .80380Achroplan 633/0.75

Air577 ,750 1.57 42.060.5 .50 .70370

C-Apo 403/1.2 W 361 ,450 0.23 21.560.5 .70 .1103110Achroplan 403/0.6 Air 722 ,800 1.8 4460.5 .60 .1003100

FIG. 3. Block diagram and schematicrepresentation of the hardware controland dissection sequence. The acquisi-tion and the irradiation protocols runin parallel. The image grabbing runsthrough an IEEE 1394 interfacewhereas the other components aredriven via the PCs RS232 ports. Theirradiation positions are first down-loaded to the scan controlling boardand then executed after the objectivepositioner has placed the focus in thesample plane.



excellent beam quality because aberrations are avoided, butis considerably slower for multiple-pulse dissection since thestage has to be moved and stabilized before irradiation. Thetime periods for single pulse shots with this solution weremeasured to be larger than 400 ms and depend on the dis-tance traveled. Furthermore, imaging of the sample duringirradiation is not possible because of the stage motion. An-other potential drawback is the stage control, which, depend-ing on the manufacturer, can become a nontrivial softwareprogramming effort.

III. PERFORMANCE

The laser-glass interaction was used to measure the dif-fraction limited UV spot dimensions. According to Snell’slaw, at the interface between two media 1 and 2 the numeri-cal aperture is conserved:

n1 sin~a1!5n2 sin~a2!5NA.

Therefore using a different medium to measure the systemaccuracy makes sense because the measured spot diameter asa function of the NA is not affected by a refractive indexchange. This assumes that the experiment is not performedtoo deep inside a glass volume, which could result in aber-rations: each objective lens is corrected only for specificworking conditions, which affect the beam spot diameter andthe irradiation efficiency. The glass patterning process is asimple method to visualize the effect of pulsed laser-inducedplasma formation and allows us to find reasonable conditionsfor processing biological tissues. As shown in Fig. 2, theglass properties are modified at the threshold of plasma for-mation with low energy pulses. At higher energies plasmashielding is likely to occur, provoking mechanical and ther-mal stress as shown in the wide glass in Fig. 2~c!. To char-acterize the strength of the plasma-induced ablation process,the plasma volume at ionization thresholdVp , equivalent tothe affected volumeVg previously measured, was comparedto the beam focal volumeVf . The obtained ratioVp /Vf

55.2 can be compared to data provided by Venugopalanet al.3 who reported plasma formation in water media with a633/0.9 objective and 6 ns pulses at 532 and 1064 nm.

Their experiments resulted in a ratioVp /Vf of 117 and 16.3at 1064 and 532 nm, respectively, at plasma threshold forma-tion and with a focal volume calculated with a Gaussianapproximation using the Rayleigh range as half height of acylinder representing the focal volume. However, with highnumerical aperture lenses~with sina.0.5!, the Gaussian ap-proximation is not valid. Moreover, considering the focalvolume to be cylindrical results in an overestimation of thevolume by 50% compared to an ellipsoid. Using formulas~1!and ~2! and an ellipsoid geometry, the same measurementslead to even higherVp /Vf ratios of 207 and 28.3 at 1064 and532 nm, respectively. Our experimentally determined ratioVp /Vf of 5.2 shows that using the frequency tripled pulsedNd:YAG laser and a high NA immersion lens improves theirradiation accuracy dramatically.

When performing intracellular surgery, one wants toavoid mechanical side effects such as cavitation or shock-wave formation. Those physical effects arise with high-energy plasmas and can result in an extended ablation vol-ume causing biological tissue damage far outside the focalvolume. Using water or a biological medium, cavitation canbe generated by the formation of bubbles or the explosion ofcell membranes due to high internal pressure caused byshockwave propagation. Both physical effects are inducedabove a threshold in irradiance ranging from about 100 to120 GW cm22. These values are similar to data fromVenugopalanet al.3 who reported thresholds of 77 and 187GW cm22 at 532 and 1064 nm, respectively. However, thepulse energy involved in cavitation and shockwave forma-tion is much lower in our case, i.e., about 0.25mJ to becompared with 1.89 and 18.3mJ at 532 and 1064 nm, respec-tively. This difference of a factor of 7–70 in pulse energyallows us to conclude that the mechanical strength of shock-wave or cavitation is reduced because much less energy istransferred to the microplasma.

In a biological application, the sample scanning versionof the setup presented here was utilized to ablate cen-trosomes in a living one-cell stageC. elegansembryo.16 Thisversion does not use galvanometric mirrors but moves thestage to irradiate different locations in the sample. Further-

FIG. 4. Timing diagram for an irradia-tion sequence.Dt1 refers to the periodfor downloading the mirrors’ positionsfor n laser pulses~Pos X0, Pos Y0,...,Pos Xn, Pos Yn!, Dt2 represents theirradiation period. The minimum pe-riod for the execution of a single shotirradiation is achieved in about 70 ms.



more, it does not contain a piezoelectric positioner: insteadthe laser beam is defocused to compensate for the chromaticaberrations of the lens, which means that the optical setup isnontelecentric. While this implementation results in a de-crease in versatility of the system and in beam quality at thefocus, we were still able to reproducibly perform centrosomedisintegration experiments with this setup.

We studied the positioning of individual microtubuleasters.16 This process is particularly important for asymmet-ric spindle positioning, where both microtubule asters areeccentrically positioned within the cell, directing the cleav-age furrow to an off-center position and allowing for thecreation of one larger and one smaller daughter cell. Thistype of asymmetric cell division is inherent to any develop-ing organism as they participate in the generation of cell fatediversity. A microtubule aster is a polar structure with astralmicrotubules emanating from the centrosome, which is at thecenter of the aster. The minus ends of microtubules are lo-cated at the centrosome, and their plus ends reach out to thecortex. A minus-end directed microtubule motor~such as dy-nein! anchored to the cortex can capture an astral microtu-bule and start to walk towards the microtubule’s minus end.It will thereby exert a pulling force upon the aster.18 As astralmicrotubules point out from the centrosome in all directions,these pulling forces determine the positioning behavior of amicrotubule aster. Now if the centrosome is disintegrated andbroken up into smaller fragments, tension is released andthese fragments start to move towards the cortex, thus allow-

ing visualization of the forces exerted upon the aster. Figure5 illustrates this effect. The anterior centrosome on the leftwas irradiated with 100 shots at 100 Hz repetition rate and;0.4 mJ/pulse~i.e., with ca. 5% peak power!. Shots wereapplied to the corners of a 2.532.5 mm rectangle, thus caus-ing the centrosome with a diameter of;3 mm to be com-pletely disintegrated. Fragments of the aster have moved outtowards the cortex. These fragments are followed and theirvelocities are measured in a live assay.16 A statistical analysisof the speeds of the fragments yields an approximation of thenumber of force generators that are available to each micro-tubule aster in a mitotic one-cell stageC. elegansembryo.16

Applications of short pulsed-laser-induced micro-plasmas, and in particular femtosecond pulsed lasers, havedeveloped quickly over the last years in optical com-munications20 and information storage.21 By focusing shortlaser pulses into glass volumes, patterns can be written andread and material properties can be modified. Working with500 ps pulses, our system offers a direct application of thisprocess in biology by hardcodingin situ information aboutsingle cells or organisms inside the glass volume of theirsupport ~coverslip!. Figure 6 illustrates this application. Asample of fission yeast cells lies at the surface of a coverslipand information is generated in the glass volume below thesample. The laser beam was focused 6mm below the cellplane and text coordinates were coded and sent to the scancontroller. Tracking back the processed samples is possiblewith this technique, as well as storing all kinds of informa-tion at the sample site~experiment parameters, date, scalebar, bar codes of various type,...!. Glass patterning is alsoused in Fig. 7 to demonstrate the three dimensional flexibil-ity of the system. This three-dimensional feature makes the

FIG. 5. A mitotic one-cell stageC. elegansembryo in which the anteriorcentrosome was disintegrated with the UV laser. The embryo contains GFPa-tubulin ~Ref. 19!. Anterior ~A! is on the left, posterior~P! is on the right.~a! Embryo prior to disintegration imaged with a spinning-disk confocalmicroscope. The arrow points to the centrosome that will be disintegrated.~b! Embryo 15 s after disintegration. Individual aster fragments~arrows!have been pulled out towards the cortex due to a release of tension at thecentrosome after UV laser ablation. Irradiation is spatially confined, as thenonirradiated centrosome and its aster remain unaffected~arrowhead!. Scalebar, 10mm.

FIG. 6. Pulsed laser-induced microplasma are used to encode informationnear the sample location, but inside the glass coverslip~6 mm below thewater/glass interface!. Observed in DIC with a condenser aperture of 0.15,the sample and the information planes are visualized simultaneously. On themiddle left of each picture, an arrow points towards the sample of interest.Below the sample, a 10mm scale bar has been inscribed.

FIG. 7. Demonstration of the 3D flexibility of the system. A pyramid ispatterned inside a glass slide by inscribing concentric squares with an axialseparation of 3mm. In ~a! and ~b!, two XY images of squares which areabout 18mm apart along the optical axis are shown.~c! XZ view extractedfrom the center of the stack.



instrument an optimal tool for developmental biology appli-cations. Thick samples can be penetrated by the UV beam aslong as the absorption and scattering of the medium are suf-ficiently low.

The combination of UV-A irradiation at a pulse width of500 ps, fast scanning, high numerical aperture, and largeworking distance lenses makes this versatile nanodissectionsetup an optimal tool to perform laser ablation with im-proved plasma confinement and very low energy levels.Delicate ablation is possible within a broad range ofin vivoapplications from intracellular nanosurgery in cell biology toembryo surgery in developmental biology. In general, thehigh level of molecular characterization in a number of bio-logical model organisms has opened the door to a furtherstudy of the mechanical details involved in processes such asspindle positioning, chromosome segregation, cleavage fur-row ingression, etc. However, to draw conclusions about me-chanics requires mechanical perturbation experiments. Thesevering of a biological structure to test for mechanical ten-sion within that structure by the use of a pulsed UV laser hasproven to be and will remain a powerful tool to illuminatethe micromechanical details involved in these complex pro-cesses.

ACKNOWLEDGMENTS

The authors wish to thank Carl Zeiss~Germany! for pro-viding the microscope apparatus, EMBL workshop engineersAlfons Riedinger, Georg Ritter, and Wolfgang Dilling fortheir contributions to the software, electronics, and mechan-ics, respectively, and finally, Dr. Jim Swoger for critical com-ments on the manuscript.

1M. W. Bernset al., Science213, 505 ~1981!.2K. O. Greulich, G. Pilarczyk, A. Hoffmann, G. M. Z. Ho¨rste, B. Scha¨fer,V. Uhl, and S. Monajembashi, J. Microsc.198, 182 ~2000!.

3V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, Phys. Rev. Lett.88,078103~2002!.

4R. Srinivasan, Science234, 559 ~1986!.5M. Niemz, Laser-Tissue Interactions. Fundamentals and Applications,2nd ed., Springer Biological and Medical Physics Series~Springer, Berlin,2002!.

6V. Venugopalan, N. S. Nishioka, and B. B. Mikic´, Biophys. J.69, 1259~1995!.

7V. Venugopalan, Proc. SPIE2391, 184 ~1995!.8B. M. Kim, M. D. Feit, A. M. Rubenchik, E. J. Joslin, P. M. Celliers, J.Eichler, and L. B. Da Silva, J. Biomed. Opt.6, 332 ~2001!.

9J. A. Izatt, D. Albagli, M. Britton, J. M. Jubas, I. Itzkan, and M. S. Feld,Lasers Surg. Med.11, 238 ~1991!.

10D. Albagli, M. Dark, L. Perelman, C. von Rosenberg, I. Itzkan, and M. S.Feld, Opt. Lett.19, 1684~1994!.

11J. Noack and A. Vogel, IEEE J. Quantum Electron.35, 1156~1999!.12A. Vogel, J. Noack, K. Nahen, D. Theisen, S. Busch, U. Parlitz, D. X.

Hammer, G. D. Noojin, B. A. Rockwell, and R. Birngruber, Appl. Phys. B:Lasers Opt.68, 271 ~1999!.

13E. Willingham, Scientist16, 42 ~2002!.14N. J. Salmon, J. E. N. Jonkman, and E. H. K. Stelzer,Proceedings of the

12th IEEE International Congress on Real Time for Nuclear and PlasmaSciences~IEEE, New York, 2001!.

15S. W. Grill, P. Gonczy, E. H. K. Stelzer, and A. A. Hyman, Nature~Lon-don! 409, 630 ~2001!.

16S. W. Grill, J. Howard, E. Scha¨ffer, E. H. K. Stelzer, and A. A. Hyman,Science301, 518 ~2003!.

17S. W. Grill and E. H. K. Stelzer, J. Opt. Soc. Am. A16, 2658~1999!.18D. J. Sharp, G. C. Rogers, and J. M. Scholey, Nature~London! 407, 41

~2000!.19K. Oegema, A. Desai, S. Rybina, M. Kirkham, and A. A. Hyman, J. Cell

Biol. 153, 1209~2001!.20M. Li, K. Mori, M. Ishizuka, X. Liu, Y. Sugimoto, N. Ikeda, and K.

Asakawa, Appl. Phys. Lett.83, 216 ~2002!.21G. Cheng, Y. Wang, J. D. White, Q. Liu, W. Zhao, and G. Chen, J. Appl.

Phys.94, 1304~2003!.



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