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Laser Microsurgery in Cell and Developmental Biology

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Laser Microsurgery in Cell and Developmental Biology adducts or of diadduct cross-linki ng in the case of laser light-stimulated binding of psoralen to nucleic acids (9). A third possibility is the generation of damage by an uncommon physical effect that occurs when ultrahigh photon densities Laser Microsurgery in Cell and are achieved in very short periods (a few nanoseconds or picoseconds). The re­ sulting nonlinear optical effects, such Developmental Biology as multiphoton absorption, dielectric breakdown, and pressure phenomena, Michael W. Berns, J. Aisl, J. Edwards, K. Strahs, J. Girton occur when the classic law of reciprocity P. McNeill, J.B. Rattner, M. Kitzes, M. Hammer-Wilson does not hold; these effects may be re­ sponsible for some of the disruption ob­ L.-H. Liaw, A. Siemens, M. Koonce, S. Peterson, S. Brenner served in biological material (10). Whiclt­ J. Burt, R. Walter, P. J. Bryant, D. van Dyk, J. Coulombe ever of the above damage-producing mechanisms is operating, whether "clas­ T. Cahill, G. S. Berns sical" or "uncommon," the damage of­ ten can be confined to a specific cellular or subcellular target in a consistent and controllable way. In addition, once the The laser microbeam permits selective system to permit exposure of living cells biophysical mechanism of laser interac­ alteration of part of a subcellular organ­ to ultrashort pulses of light. This dual tion with the molecules is ascertained, elle in a single living cell. This alteration laser system is interfaced with an invert­ the investigator has a method for precise can be in a specific class of molecules ed Zeiss Axiomat microscope and an disruption of a specific class of mole­ confined to an area of less than 0.25 image array processing computer, which cules within a strictly delimited region of micrometer. This capability has been de­ permits the exposure of groups of cells, the living cell. The size of this region veloped over the last 15 years and is now single cells, or individual organelles may be considerably smaller than the generally available for studies in cell and within single cells to a variety of wave­ size of the focused laser beam because of developmental biology. lengths at various power densities, with the distribution of the target molecules in the target zone. However, the size of the focused laser spot also is of paramount Summary. New applications of laser microbeam irradiation to cell and developmen­ importance, because it defines the maxi­ tal biology include a new instrument with a tunable wavelength (217- to 800- mum volume of biological material that nanometer) laser microbeam and a wide range of energies and exposure durations will be available for direct interaction (down to 25 x 10- 12 second). Laser microbeams can be used for microirradiation of with the laser photons. Though the diam­ selected nucleolar genetic regions and for laser microdissection of mitotic and eter of the focused laser spot is a direct cytoplasmic organelles. They are also used to disrupt the developing neurosensory function of the wavelength, the magnifi­ appendages of the cricket and the imaginal discs of Drosophila. cation of the focusing objective, and the numerical aperture of the objective, the actual diameter of the "effective" lesion Just over 12 years ago, the blue-green time exposures as short as 25 psec. In area may be considerably less than the argon ion laser microbeam was intro­ addition, the use of the sophisticated theoretical limit of the focused laser duced as a potential tool for subcellular Zeiss Axiomat microscope and the im­ beam, which is half the wavelength. This microsurgery (/, 2). There had been lim­ age processing computer permits a state­ is because a high-quality laser beam can ited success earlier with the red ruby of-the-art optical and photometric ex­ be generted in the transverse electro­ laser (3) and the classical ultraviolet mi­ amination of the biological material. magnetic (TEM00) mode, which results crobeam (4). The work with the blue­ Since this user system is now available in a beam with a Gaussian energy profile green argon laser led to development of a to the scientific community (7), we will across it. The profile is carried over to tunable wavelength flash-lamp-pumped review some of the earlier key experi­ the focused spot, producing a " hot spot" dye laser microbeam (5) and later to a ments and several recent unpublished of energy in the center. It has been dye laser that was pumped by the green experiments that demonstrate the use demonstrated consistently (11) that by wavelength of a low-power neodymium­ and versatility of the system in cell and careful attenuation of the raw laser y AG (yttrium-aluminum-garnet) laser developmental biology. beam, the damage-producing portion in (6). The recent establishment of a Na­ tional Institutes of Health Biotechnology Michael W. Berns is in the Department of Devel­ "user" resource has permitted the de­ Principles of Selective Damage opmental and Cell Biology, University of California. Irvine 92717; J. Aist is in the Department of Plant velopment of a dye laser microbeam Pathology, Cornell University, Ithaca. New York completely tunable from 217 to 800 nano­ Laser light is intense, coherent, mono­ 14853; J. Edwards is in the Department of Zoology. University of Washington, Seattle 98195; K. Strahs meters, by employing the second (532 chromatic electromagnetic radiation. is affiliated with Beckman Instruments, Inc .• Fuller· nm), third (355 nm), and fourth (265 nm) The damage produced by a focused laser ton. California 92632 ; J. Girton is a member of the School of Life Sciences, University of Nebraska, harmonic wavelengths of a high-power beam may be due to classical absorption Lincoln 68588; P. McNeill, J.B. Rattner, M. Kitzes, I0-nanosecond pulsed neodymium-Y AG by natural or applied chromophores and M. Hammer-Wilson, L.-H. Liaw, A. Siemens. M. Koonce, S. Peterson, S. Brenner, J. Burt, R. Wal· laser (Fig. I). In addition, a separate the subsequent generation of heat (8), or ter, P. J. Bryant , D. van Dyk, J. Coulombe, T. high-power 25-picosecond neodymium­ Cahill, and G. S. Berns are affiliated with the De· it may be caused by a photochemical partmcnt of Developmental and Cell Biology, Uni· y AG laser has been integrated into the process such as the production of mono- versity of California, Irvine 92717. SCIENCE. VOL. 213. 31JULY1981 0036-807518110731-0505$02.00/0 Copyright 10 1981 AAAS sos the focused spot can be confined to the result, lesions can be routinely produced Video Computer Microscopy central hot spot (that is, the only region that are less than 0.25 µm in diameter within the focused spot that is above the and frequently 0.1 µm in diameter (see Since the laser beam can be focused to threshold for damage production). As a cover). produce a damage spot less than the diffraction limits of the light microscope, one of the limiting factors becomes the Monitoring, ottenuotion, quality of the optical image itself. It is filtering devices difficult lo focus the laser beam onto a target that is not readily visible. 5 266 We have been able to improve upon ""' ~·~· nm Nd-YAG laser the optical image of the highest quality 7 109 w 25x 10-12 sec. (picoseconds) light microscope (the Zeiss Axiomat) by 355 26 ~~· • ~A- Nd-YAG laser using a low light level television camera 7 lrl> W ~ 15 x 10-9 sec. (nanoseconds) (Newvicon tube) whose signal is digi­ tized and enhanced by a fast (real time) ~ -~-700~m- -== - Dye laser image-processing computer. Recent ad­ ' 105w vances in computer technology have led ....--::::-....,..,/ 0.5 J'm Dioitol X-Y Stoot to the development of small, relatively inexpensive image array processprs that Dioilizer LSI 11/2 Oeonzo lmooe are capable of performing sophlsticatd Minicomputor, Array Processor Ouol Floppy Discs (Gray Sc;ole) image-processing routines on video im­ Inverted ages in real time. Real-time processing Zeiss Axiomot @@ CRT allows sophisticated routines, such as DIC, Pol., Monitor Video Timt Phase, Quortz Microscope Lapse Tope I~ [D]l contrast enhancement, edge detection, Recorder . background subtraction, multiple image Color Monllor averaging, and pseudocolor enhance­ Fig. I. Diagram of laser microbeam system. The three basic components of the system are the ment; all of these can be performed on lasers (Quante! Y AG 400 and 481ffDL III), the microscope (Zeiss inverted Axiomat equipped the microscope image during the time of for phase contrast, bright field , polarization, and differential interference contrast), and the television computer system (DcAnza IP 5000 image array processor, Sierra LST-1 television the actual experiment. Figure 2 and the camera, and GYYR DA 5300 MKIII videotape system). An LSI-I I minicomputer is used to cover photograph are examples of com­ drive the image array processor. In addition, the image processor-LSI combination is puter-enhanced images. The processor interfaced to the X-Y digital microscope stage in order to provide cell tracking capabilities. can also be used for more analytical tasks such as calculation of object areas, boundary lengths, and intracellar dis­ tances. The integration of the computer into the laser microbeam system has greatly extended the capabilities of this system and the scope of the experiments to be described. Chromosome Microsurgery In 1969, a low-power pulsed argon ion laser was focused on chromosomes of living mitotic salamander ceUs that had been photosensitized with the vital dye acridine orange. The result was the pro­ duction of a 0.5-µ.m lesion in the irradiat­ ed region of the chromosome (Fig. 3). Subsequent studies on salamander and rat kangaroo cells (PTK1 and PTK2) demonstrated that the laser microbeam could be used to selectively inactivate a specific genetic site, the nucleolar genes (1 , /2).
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