REVIEW OF SCIENTIFIC INSTRUMENTS

VOLUME 75, NUMBER 2

FEBRUARY 2004

Ultraviolet diffraction limited nanosurgery of live biological tissues

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

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

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

Received 25 September 2003; accepted 15 October 2003 A laser nanodissection system for in vivo and in situ biological tissues is presented. A pulsed laser beam operating at a wavelength of 355 nm enables diffraction limited dissection, providing an optimal tool for intracellular nanosurgery. Coupled into a conventional inverted microscope and scanned across a eld of up to 100 100 m2 , this optical nanoscalpel performs in vivo photoablation and plasma-induced ablation inside organisms ranging from intracellular organelles to embryos. The system allows the use of conventional microscopy contrasts and methods, fast dissection with up to 1000 shots per second, and simultaneous dissection and imaging. This article outlines an efcient implementation with a small number of components and reports an improvement 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 of writing information directly at the sample location by plasma glass nanopatterning. 2004 American Institute of Physics. DOI: 10.1063/1.1641163

I. INTRODUCTION

Laser microsurgery of biological tissues13 has been studied for over 30 years but is still a eld of thorough research and major controversies. The mechanisms of lasertissue interactions have been extensively investigated.310 The underlying physical principles are photoablation and plasma-induced ablation. Photoablation4 6 occurs by means of photochemical reactions. The energy of UV photons is sufciently high typically 3.5 eV to reach the molecular dissociation energy and, therefore, to break chemical bonds. This leads to the photodecomposition of the irradiated molecules. For biological applications, the most suitable wavelengths in this respect are in the UV-A range 315 nm 400 nm , since UV-B 280 nm 315 nm is strongly absorbed by DNA and provokes irreversible damage and mutations in living samples. UV-C 100 nm 280 nm absorption is too high in turbid media and would also require special optical elements, thus raising the cost and decreasing the exibility of an irradiation setup based on a conventional microscope. Plasma formation occurs above a certain energy or power density threshold3,5,11,12 when ionization of the medium starts by means of thermal or multiphoton ionization. Ablation occurs inside the plasma volume, the shape and size of which depend on the beam geometry. The energy density threshold for ionization has been found to depend on the pulse duration.11,12 Below the ns range, the threshold in energy density decreases and microplasmas can be induced with signicantly less energy than with the nanosecond
a

Electronic mail: colombel@embl.de 472

pulses commonly utilized in commercial microdissection systems.13 This results in delicate ablation, i.e., mechanical side effects like shockwave or cavitation, likely to damage the tissue structure far outside the ablating spot area, can be reduced or avoided. With this in mind, a nanodissection system was implemented on a conventional inverted microscope. Using a pulsed UV laser with sub-ns pulse width and a high numerical aperture NA lens, low energy ablation due to the short pulse in a highly conned volume due to the short wavelength of 355 nm is possible. Peak power densities of up to 10 TW cm 2 are achieved with a low energy laser source 8.8 J per pulse . Potential mechanical or thermal damage to the living samples outside the focal volume are avoided during this ablation process. The setup combines features from available commercial systems and overcomes many of their limitations. In particular, we achieve diffraction limited focusing with short sub-ns ablation avoiding severe thermal and mechanical damages to living samples , fast beam scanning, and optimal coupling. This allows simultaneous in vivo dissection and image acquisition. All microscopy modes remain available, including uorescence and a port for a confocal module. The working distances of available microscope objective lenses allows one to perform subcellular surgery as well as thick sample dissection e.g., in living embryos . Here, we present a detailed description of this device, discuss the technical accuracy, speed, and potential biological applications, and present the option to store information directly at the sample site by means of plasma-induced glass patterning inside the sample holder e.g., cover slip .
2004 American Institute of Physics

0034-6748/2004/75(2)/472/7/$22.00

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Rev. Sci. Instrum., Vol. 75, No. 2, February 2004

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FIG. 1. Two schematic representations a and b and a photograph c of the UV nanoirradiation setup, the second scheme b shows the optical path from above. The power of the frequency tripled Nd:YAG laser 1 is controlled by an AOTF 2 . The beam is expanded by a telecentric UV lens 3, magnication 3 , hits a galvomirror pair 4 and is coupled into an Axiovert 200M microscope 10 with two telecentric UV singlet lenses 5, resulting magnication factor 2 and a dichroic mirror mounted on a slider 7 at the eld aperture location. Arrows indicate the orientation of the laser lights polarization. The system transmission is optimized using an s-polarized beam hitting the uorescence beam splitter 8 . Simultaneous dissection and uorescence image acquisition is possible by placing the excitation lters at the back of the uorescence port in a lter wheel 6 . Other elements: 9 objective piezoelectric positioner, 11 DSP board and the galvanometers analog drivers, 12 uorescence lamp shutter.

II. INSTRUMENT A. Overview

The instrument is based on a Zeiss inverted microscope Axiovert 200M as shown in Fig. 1. No major hardware modication is necessary to achieve a high performance setup. The main parts are listed in Table I together with their specications. In the setup, a pulsed UV laser Nd:YAG at 355 nm third harmonic of D 1064 nm) is expanded by means of telecentric optics to meet the diffraction limit with a Zeiss 63 /1.2W lens. The theoretical beam diameter in the focal plane is 361 nm. A galvanometer pair scan unit guides the laser beam across a eld of 100 100 m2 . Coupling

into the microscope is achieved via the uorescence illumination path. The eld aperture slider has been modied and contains a dichroic mirror reecting at the laser wavelength of 355 nm. The laser power is controlled by an acoustooptical tunable lter AOTF . Because most objective lenses are not designed for UV-A illumination, chromatic aberrations are expected and will cause the UV laser focus to be shifted axially relative to the visible light focus. These focal shifts can be corrected by up to 100 m using a piezoelectric positioner. Both the AOTF and the piezoelectric positioner are driven via the computers RS232 interface communicating with a Scanning Controller DSP board triggering the la-

TABLE I. Main components used in the nanodissection setup with their corresponding manufacturer and main specications. Manufacturer part number JDS Uniphase Power Chip PNV-001025-050 AA optoelectronique AOTF.4C-UV Sill Optics S6ASS3103/075 GSI Lumonics XY10 Physik Instrumente PIFOC

Part Pulsed Nd:YAG laser UV Acousto-optical tunable lter AOTF UV beam expander Galvanometer mirrors Piezoelectric objective positioner
a

Specication 355 nm, energy per pulse 10 J, pulse width 500 ps, repetition rate 1 kHz Transmission at 355 nm: 92% Magnication 3 Optical scan angle 175 mrad, position accuracy 50 rad,a repeatability 10 rada Positioning range 100 m, traveling time 1 ms

Values specied by the manufacturer.

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ser pulses and synchronizing them with the galvo-mirror steps. Finally, we provide a user interface by the means of a graphical software application embedded in the CCC software.14 To control the irradiation sequence, the user may control the following parameters: pulse energy up to 8.8 J , number of pulses, repetition rate up to 1 kHz , and focus z-position correction up to 100 m . A custom target shape is dened on a live window across the full irradiation eld, and the scan controller board converts the graphical coordinates to angular coordinates that position the laser beam pulses inside the sample. The setup offers all commonly used microscopy contrast modes uorescence, differential interference contrast or DIC, phase contrast as well as confocal microscopy in vivo and also permits the use of different objective lenses.
B. Coupling and scanning the UV beam

FIG. 2. Visible effect of pulsed UV laser-glass interaction. Above a certain peak power density threshold a and b of ca. 65 GW cm 2, glass properties change inside the focal volume of the focused beam and the irradiation volume becomes visible. At higher powers in c , mechanical side effects induced by photodisruption provoke glass fracture. Images were taken with bright-eld transmission, therefore the dark and light zones do not provide direct information about the material property but only represent the reection of light by the modied glass material. b The image was extracted from a stack of X/Y images recorded along the optical axis. Scale bar 5 m.

The coupling of UV-A light into a conventional microscope via the uorescence path does not interfere with the available optical contrasts. In order to allow simultaneous dissection and imaging, the eld aperture slider of the microscope contains a dichroic mirror with a high reectance at 355 nm and high transmittance in the visible spectrum. For standard epiuorescence the classical excitation lines in the visible spectrum must be efciently transmitted: in our case, 70% transmission at 400 nm Optosigma, USA and more than 95% from 450 to 700 nm. Excitation lters are removed from the lter block and inserted into a lter wheel between the arc lamp and the microscope chassis to allow the propagation of UV laser light from the eld aperture to the objective lens. Using a Zeiss C-Apo 1.2W lens, diffraction limited focussing is achieved with a 3-times beam expander and scanning lenses magnication 2 , leading to a scan eld of 80 80 m2 with an optical scan angle of 7.5 131 mrad at the galvanometric mirrors . The accuracy and repeatability of the scanning mirrors depend mainly on the electronics. We achieve a positioning accuracy of 10 rad corresponding to 6 nm in sample plane with 16-bit digital control and stable analog drivers. The thermal noise of the electronics results in a steering accuracy of 50 rad 30 nm . Thus the positioning accuracy is signicantly better than the beam diameter in the focal plane by an expected factor of 60 . In a previous version of this setup,15,16 the microscope stage was controlled to move the sample relative to the beam. This solution has the advantage that the beam remains steady at the center position of the eld and is therefore less likely to suffer from spherical aberrations. Moreover, with this type of sample scanning, irradiation can also be performed across a larger eld of view. In our setup, we achieve diffraction limited focusing using a Zeiss C-Apo 63 /1.2W water immersion lens. A simple method for measuring the beam dimensions in the object plane is illustrated in Fig. 2. The UV beam was focused inside a conventional glass coverslip and the visible effect of one emitted pulse is observed in brighteld transmission mode for two different optical powers. The minimum detectable effect occurred with a pulse energy of 0.4 0.1 J deposited in the medium, or 65 GW cm 2 of peak

power density threshold. Figures 2 a and 2 b show a spot with a diameter d xy 0.45 0.05 m in the XY plane and a length of l z 2.50 0.25 m along Z. Figure 2 c shows the effect of a pulse with the maximum available energy, 8.8 0.1 J: a glass fracture extruding over several microns is produced. To characterize the beam focus quality, the measurements at 0.4 J can be compared to theoretical values. To estimate the dimensions of the focal volume, we consider it to be an ellipsoid with lateral extent D xy and elongation L z . The formulas appropriate for high NA lenses:17 D xy 2 /n 1 cos and L z 2 /n 3 2 cos cos 2
1/2

where is the angle used in the denition of the numerical aperture NA n sin , and n is the refractive index of the D 2 L z /6 of the sample medium. The resulting volume V f xy 3 focal ellipsoid is V f 0.050 m , and can be compared to the affected measured volume in glass V g 0.265 m3 . We therefore characterize the beam quality with the factor V g /V f 5.2. Table II lists the accuracy and working conditions of the system with different Zeiss objective lenses that have a transmission of at least 50% at 355 nm, in order to minimize the UV losses. The differences in focal extent are directly due to differences in numerical aperture of the focused beams. Using a NA 0.6 air lens instead of a NA 1.2 water immersion lens, the size of the focus is increased by a factor of 2, but the working distance is improved by seven times. Finally, in order to improve the coupling of the 355 nm laser line into the microscope and to optimize the system transmission, the beam polarization must be considered. Conventional lter sets are not designed to t a special requirement at 355 nm so their polarization dependence can be quite complicated. In general, the beam splitters reectivity is higher with an incident s-polarized beam on the beam splitter. In Fig. 1, the arrows represent the orientation of the UV beams polarization, and the coupling and scanning optical elements are arranged to achieve an s-polarized beam. Nevertheless, standard commercial lters will not always reect sufcient power to perform nanosurgery. Two solutions can be used to achieve high reectance. First, the beam splitter can be coated with a special layer that reects the 355 nm

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Rev. Sci. Instrum., Vol. 75, No. 2, February 2004

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TABLE II. Different objective lenses used for 355 nm nanodissection system. Diffraction limited focusing is achieved under particular conditions with high NA 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 thick samples applications. Theoretical Airy disk nm 361 577 361 722 Measured X-Y spot accuracy nm 450 750 450 800 Free working distance mm 0.24 1.57 0.23 1.8 Focus aberration at 355 nm m 4.0 0.5 42.0 0.5 21.5 0.5 44 0.5 Specied transmission % 50 50 70 60 Measured scanning eld m 80 80 70 70 110 110 100 100

Objective lens Zeiss C-Apo 63 /1.2 W Achroplan 63 /0.75 Air C-Apo 40 /1.2 W Achroplan 40 /0.6 Air

line. Second, one can use double-band lter sets specied for two chromophores, one in the near UV such as 4 ,6Diamidino-2-phenylindole or DAPI , to ensure high reectance of the beam splitter, and the other for the chromophore of interest. In our setup, a maximum laser power in the focus can reach up to 40% of the total output laser power, since we use a beam splitter optimized for the 355 nm line and an objective lens with high transmission. This means that up to 8.8 J can be deposited in the sample per pulse, corresponding to an estimated peak power density of about 10 TW cm 2 with a diffraction limited beam.
C. Synchronization and software

The software interface of the system is based on the software for the Compact Confocal Camera.14 Users outline a target on a live image and control laser output power, number of laser pulses, objective defocus, and mirror pair scanning speeds. The dynamic behavior of the system is limited by the optimal laser repetition rate of 1 kHz. Any point in the scan eld can thus be reached and irradiated once every millisecond. The digital controller for the mirrors, the AOTF, and the piezoelectric positioner are controlled via the RS232 interface of a PC. The standard features of the microscope operate independently of the irradiation protocol, i.e., its hardware does not have to be controlled. In Fig. 3, a block diagram shows the logical sequence of a typical irradiation procedure together with the hardware

connections. Figure 4 presents the timing structure of this sequence. After the user has set the parameters for dissection, target, and the acquisition e.g., how many images before and after the irradiation are captured , the nanosurgery process is started. The irradiated positions are downloaded to the Scan Controller board SC2000 at a speed of 115.2 kB/s and are converted to angle values for the scan mirrors. The duration of this download was measured to last t1 (6.1n 48) ms, where n is the number of shots. The piezoelectric objective positioner then places the UV beam focus in the image plane i.e., the chromatic shift of the UV focus is compensated . The surgical sequence is executed by the SC2000, which synchronizes the laser pulses with the mirrors steps at a frequency f up to 1 kHz, and therefore lasts a time period of t1 n/ f . The objective positioner is then moved back so that the image plane is in focus. The total time interval between the irradiation command and the end of the dissection procedure is t (6.1n n/ f 63) ms. For example, the whole protocol for irradiating a hundred pulses along a line at 1 kHz would last 773 ms, with only 10 ms being used for the optical dissection sequence. A single pulse irradiation is achieved in about 70 ms. The acquisition runs in parallel with an IEEE 1394 charge coupled device camera, triggering a shutter for uorescence mode as shown in Fig. 3. One alternative to this beam-scanning system is to directly drive the stage of the microscope in order to move the sample relative to a xed beam. This solution always offers

FIG. 3. Block diagram and schematic representation of the hardware control and dissection sequence. The acquisition and the irradiation protocols run in parallel. The image grabbing runs through an IEEE 1394 interface whereas the other components are driven via the PCs RS232 ports. The irradiation positions are rst downloaded to the scan controlling board and then executed after the objective positioner has placed the focus in the sample plane.

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FIG. 4. Timing diagram for an irradiation sequence. t1 refers to the period for downloading the mirrors positions for n laser pulses Pos X0, Pos Y0,..., Pos Xn, Pos Yn , t2 represents the irradiation period. The minimum period for the execution of a single shot irradiation is achieved in about 70 ms.

excellent beam quality because aberrations are avoided, but is considerably slower for multiple-pulse dissection since the stage has to be moved and stabilized before irradiation. The time periods for single pulse shots with this solution were measured to be larger than 400 ms and depend on the distance traveled. Furthermore, imaging of the sample during irradiation is not possible because of the stage motion. Another potential drawback is the stage control, which, depending on the manufacturer, can become a nontrivial software programming effort.
III. PERFORMANCE

The laser-glass interaction was used to measure the diffraction limited UV spot dimensions. According to Snells law, at the interface between two media 1 and 2 the numerical aperture is conserved: n 1 sin
1

n 2 sin

NA.

Therefore using a different medium to measure the system accuracy makes sense because the measured spot diameter as a function of the NA is not affected by a refractive index change. This assumes that the experiment is not performed too deep inside a glass volume, which could result in aberrations: each objective lens is corrected only for specic working conditions, which affect the beam spot diameter and the irradiation efciency. The glass patterning process is a simple method to visualize the effect of pulsed laser-induced plasma formation and allows us to nd reasonable conditions for processing biological tissues. As shown in Fig. 2, the glass properties are modied at the threshold of plasma formation with low energy pulses. At higher energies plasma shielding is likely to occur, provoking mechanical and thermal stress as shown in the wide glass in Fig. 2 c . To characterize the strength of the plasma-induced ablation process, the plasma volume at ionization threshold V p , equivalent to the affected volume V g previously measured, was compared to the beam focal volume V f . The obtained ratio V p /V f 5.2 can be compared to data provided by Venugopalan et al.3 who reported plasma formation in water media with a 63 /0.9 objective and 6 ns pulses at 532 and 1064 nm.

Their experiments resulted in a ratio V p /V f of 117 and 16.3 at 1064 and 532 nm, respectively, at plasma threshold formation and with a focal volume calculated with a Gaussian approximation using the Rayleigh range as half height of a cylinder representing the focal volume. However, with high 0.5 , the Gaussian apnumerical aperture lenses with sin proximation is not valid. Moreover, considering the focal volume to be cylindrical results in an overestimation of the volume by 50% compared to an ellipsoid. Using formulas 1 and 2 and an ellipsoid geometry, the same measurements lead to even higher V p /V f ratios of 207 and 28.3 at 1064 and 532 nm, respectively. Our experimentally determined ratio V p /V f of 5.2 shows that using the frequency tripled pulsed Nd:YAG laser and a high NA immersion lens improves the irradiation accuracy dramatically. When performing intracellular surgery, one wants to avoid mechanical side effects such as cavitation or shockwave formation. Those physical effects arise with highenergy plasmas and can result in an extended ablation volume causing biological tissue damage far outside the focal volume. Using water or a biological medium, cavitation can be generated by the formation of bubbles or the explosion of cell membranes due to high internal pressure caused by shockwave propagation. Both physical effects are induced above a threshold in irradiance ranging from about 100 to 120 GW cm 2. These values are similar to data from Venugopalan et al.3 who reported thresholds of 77 and 187 GW cm 2 at 532 and 1064 nm, respectively. However, the pulse energy involved in cavitation and shockwave formation is much lower in our case, i.e., about 0.25 J to be compared with 1.89 and 18.3 J at 532 and 1064 nm, respectively. This difference of a factor of 770 in pulse energy allows us to conclude that the mechanical strength of shockwave or cavitation is reduced because much less energy is transferred to the microplasma. In a biological application, the sample scanning version of the setup presented here was utilized to ablate centrosomes in a living one-cell stage C. elegans embryo.16 This version does not use galvanometric mirrors but moves the stage to irradiate different locations in the sample. Further-

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

FIG. 5. A mitotic one-cell stage C. elegans embryo in which the anterior centrosome was disintegrated with the UV laser. The embryo contains GFP -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 confocal microscope. 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 the centrosome after UV laser ablation. Irradiation is spatially conned, as the nonirradiated centrosome and its aster remain unaffected arrowhead . Scale bar, 10 m.

more, it does not contain a piezoelectric positioner: instead the laser beam is defocused to compensate for the chromatic aberrations of the lens, which means that the optical setup is nontelecentric. While this implementation results in a decrease in versatility of the system and in beam quality at the focus, we were still able to reproducibly perform centrosome disintegration experiments with this setup. We studied the positioning of individual microtubule asters.16 This process is particularly important for asymmetric spindle positioning, where both microtubule asters are eccentrically positioned within the cell, directing the cleavage furrow to an off-center position and allowing for the creation of one larger and one smaller daughter cell. This type of asymmetric cell division is inherent to any developing organism as they participate in the generation of cell fate diversity. A microtubule aster is a polar structure with astral microtubules emanating from the centrosome, which is at the center of the aster. The minus ends of microtubules are located at the centrosome, and their plus ends reach out to the cortex. A minus-end directed microtubule motor such as dynein anchored to the cortex can capture an astral microtubule and start to walk towards the microtubules minus end. It will thereby exert a pulling force upon the aster.18 As astral microtubules point out from the centrosome in all directions, these pulling forces determine the positioning behavior of a microtubule aster. Now if the centrosome is disintegrated and broken up into smaller fragments, tension is released and these fragments start to move towards the cortex, thus allow-

ing visualization of the forces exerted upon the aster. Figure 5 illustrates this effect. The anterior centrosome on the left was irradiated with 100 shots at 100 Hz repetition rate and 0.4 J/pulse i.e., with ca. 5% peak power . Shots were applied to the corners of a 2.5 2.5 m rectangle, thus causing the centrosome with a diameter of 3 m to be completely disintegrated. Fragments of the aster have moved out towards the cortex. These fragments are followed and their velocities are measured in a live assay.16 A statistical analysis of the speeds of the fragments yields an approximation of the number of force generators that are available to each microtubule aster in a mitotic one-cell stage C. elegans embryo.16 Applications of short pulsed-laser-induced microplasmas, and in particular femtosecond pulsed lasers, have developed quickly over the last years in optical communications20 and information storage.21 By focusing short laser pulses into glass volumes, patterns can be written and read and material properties can be modied. Working with 500 ps pulses, our system offers a direct application of this process in biology by hardcoding in situ information about single cells or organisms inside the glass volume of their support coverslip . Figure 6 illustrates this application. A sample of ssion yeast cells lies at the surface of a coverslip and information is generated in the glass volume below the sample. The laser beam was focused 6 m below the cell plane and text coordinates were coded and sent to the scan controller. Tracking back the processed samples is possible with this technique, as well as storing all kinds of information at the sample site experiment parameters, date, scale bar, bar codes of various type,... . Glass patterning is also used in Fig. 7 to demonstrate the three dimensional exibility of the system. This three-dimensional feature makes the

FIG. 7. Demonstration of the 3D exibility of the system. A pyramid is patterned inside a glass slide by inscribing concentric squares with an axial separation of 3 m. In a and b , two XY images of squares which are about 18 m apart along the optical axis are shown. c XZ view extracted from the center of the stack.

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1 2

Colombelli, Grill, and Stelzer M. W. Berns et al., Science 213, 505 1981 . K. O. Greulich, G. Pilarczyk, A. Hoffmann, G. M. Z. Horste, B. Schafer, V. Uhl, and S. Monajembashi, J. Microsc. 198, 182 2000 . 3 V. Venugopalan, A. Guerra, K. Nahen, and A. Vogel, Phys. Rev. Lett. 88, 078103 2002 . 4 R. Srinivasan, Science 234, 559 1986 . 5 M. Niemz, Laser-Tissue Interactions. Fundamentals and Applications, 2nd ed., Springer Biological and Medical Physics Series Springer, Berlin, 2002 . 6 V. Venugopalan, N. S. Nishioka, and B. B. Mikic, Biophys. J. 69, 1259 1995 . 7 V. Venugopalan, Proc. SPIE 2391, 184 1995 . 8 B. 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 . 9 J. A. Izatt, D. Albagli, M. Britton, J. M. Jubas, I. Itzkan, and M. S. Feld, Lasers Surg. Med. 11, 238 1991 . 10 D. Albagli, M. Dark, L. Perelman, C. von Rosenberg, I. Itzkan, and M. S. Feld, Opt. Lett. 19, 1684 1994 . 11 J. Noack and A. Vogel, IEEE J. Quantum Electron. 35, 1156 1999 . 12 A. 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 . 13 E. Willingham, Scientist 16, 42 2002 . 14 N. J. Salmon, J. E. N. Jonkman, and E. H. K. Stelzer, Proceedings of the 12th IEEE International Congress on Real Time for Nuclear and Plasma Sciences IEEE, New York, 2001 . 15 S. W. Grill, P. Gonczy, E. H. K. Stelzer, and A. A. Hyman, Nature London 409, 630 2001 . 16 S. W. Grill, J. Howard, E. Schaffer, E. H. K. Stelzer, and A. A. Hyman, Science 301, 518 2003 . 17 S. W. Grill and E. H. K. Stelzer, J. Opt. Soc. Am. A 16, 2658 1999 . 18 D. J. Sharp, G. C. Rogers, and J. M. Scholey, Nature London 407, 41 2000 . 19 K. Oegema, A. Desai, S. Rybina, M. Kirkham, and A. A. Hyman, J. Cell Biol. 153, 1209 2001 . 20 M. Li, K. Mori, M. Ishizuka, X. Liu, Y. Sugimoto, N. Ikeda, and K. Asakawa, Appl. Phys. Lett. 83, 216 2002 . 21 G. Cheng, Y. Wang, J. D. White, Q. Liu, W. Zhao, and G. Chen, J. Appl. Phys. 94, 1304 2003 .

instrument an optimal tool for developmental biology applications. Thick samples can be penetrated by the UV beam as long as the absorption and scattering of the medium are sufciently low. The combination of UV-A irradiation at a pulse width of 500 ps, fast scanning, high numerical aperture, and large working distance lenses makes this versatile nanodissection setup an optimal tool to perform laser ablation with improved plasma connement and very low energy levels. Delicate ablation is possible within a broad range of in vivo applications from intracellular nanosurgery in cell biology to embryo surgery in developmental biology. In general, the high level of molecular characterization in a number of biological model organisms has opened the door to a further study of the mechanical details involved in processes such as spindle positioning, chromosome segregation, cleavage furrow ingression, etc. However, to draw conclusions about mechanics requires mechanical perturbation experiments. The severing of a biological structure to test for mechanical tension within that structure by the use of a pulsed UV laser has proven to be and will remain a powerful tool to illuminate the micromechanical details involved in these complex processes.
ACKNOWLEDGMENTS

The authors wish to thank Carl Zeiss Germany for providing the microscope apparatus, EMBL workshop engineers Alfons Riedinger, Georg Ritter, and Wolfgang Dilling for their contributions to the software, electronics, and mechanics, respectively, and nally, Dr. Jim Swoger for critical comments on the manuscript.

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