Laser Microirradiation and Computer Video Optical Microscopy in Cell Analysis
Laser light is intense, coherent, monochromatic electromagnetic radiation. Because of these properties it can be a unique probe of cellular structure and function. The damage produced by a focused laser beam may be caused by classical absorption by natural or applied chromophores and the subsequent generation of heat (Berns and Salet, 1972), or it may be caused by a photochemical process. An example of such a process would be the production of monoadducts or diadduct cross-linking in the case of laser light’s stimulated binding of psoralens to nucleic acids (Peterson and Berns, 1978a). However, a third possibility is the generation of damage by an uncommon physical effect that occurs when ultra-high photon densities are achieved in very short periods of time (a few nanoseconds or picoseconds). The resulting nonlinear optical effects such as multiphoton absorption occur when the classic law of reciprocity does not hold. These effects may be responsible for some of the disruption observed in biological material (Berns, 1976). Whichever of the above damage-producing mechanisms is operating, be it “classical” or “uncommon,” the damage often can be confined to a specific cellular or subcellular target in a consistent and controllable way. In addition, once the biophysical mechanism of laser interaction with the molecules is ascertained, the investigator has a method for precise disruption of a specific class of molecules within a strictly delimited region of the living cell. The size of this region may be considerably smaller than the size of the focused laser beam because of the distribution of the target molecules in the target zone. However, the size of the focused laser spot also is of paramount importance because it defines the maximum volume of biological material that will be available for direct interaction with the laser photons. Though the diameter of the focused laser spot is a direct function of the wavelength, the magnification of the focusing objective, and the numerical aperture of the objective, the actual diameter of the “effective” lesion area may be considerably less than the theoretical limit of the focused laser beam. This is because a high-quality laser beam can be generated in the TEM∞ mode, which results in a beam with a gaussian energy profile across it. The profile is carried over to the focused spot, which results in a “hot spot” of energy in the center. It has been demonstrated consistently (Berns, 1974a) that by careful attenuation of the raw laser beam, the damage-producing portion in the focused spot can be confined to the central hot spot (e.g., that is the only region within the focused spot that is above the threshold for damage production). As a result, lesions can be routinely produced less than 0.25 μm in diameter, and frequently down to 0.1 μm in diameter.
KeywordsAcridine Orange Nucleolar Organizer Focus Laser Beam Functional Kinetochore Focus Laser Spot
Adkisson, K. P., Baic, D., Burgott, S., Cheng, W. K., and Berns, M. W. (1973) Argon laser microirradiation of mitochondria in rat myocardial cells in tissue culture. IV. Ultrastructural and cytochemical analysis of minimal lesions, J. Mol. Cell. Cardiol.
Allen, R. D., Allen, N. S., and Travis, J. L. (1981a) Video-enhanced contrast, differential interference contrast (AVEC-DIC) microscopy: New methods capable of analyzing microtubulerelated motility in the reticulopodial network of Allogromia iaticollaris
, Cell Motility
Allen, R. D., Travis, J. L., Allen, N. S., and Yilmaz, H. (1981b) Video-enhanced contrast polarization (AVEC-POL) microscopy: A new method applied to the detection of birefringence in the motile reticulopodial network of Allogromia iaticollaris
, Cell Motility
Berns, M. W. (1972) Partial cell irradiation with a tunable organic dye laser, Nature (London)
Berns, M. W. (1974a) Biological Microirradiation
(Biological Techniques Series), Prentice-Hall, New York.Google Scholar
Berns, M. W. (1974b) Directed chromosome loss by laser microirradiation, Science
Berns, M. W. (1975) Dissecting the Cell with a Laser Microbeam, in, Lasers in Physical Chemistry and Biophysics
(J. Joussot-Dubien, eds.), Elsevier, New York, pp. 389–401.Google Scholar
Berns, M. W. (1976) A possible two-photon effect in vitro
using a focused laser beam, Biophys. J.
Berns, M. W., and Cheng, W. K. (1971) Are chromosome secondary constrictions nucleolar organizers? A re-evaluation using a laser microbeam, Exp. Cell Res.
Berns, M. W., and Floyd, A. D. (1971) Chromosome microdissection by laser: A functional cytochemical analysis, Exp. Cell Res.
Berns, M. W., and Richardson, S. M. (1977) Continuation of mitosis after selective laser microbeam destruction of the centriolar region, J. Cell Biol.
Berns, M. W., and Rounds, D. E. (1970) Cell surgery by laser, Sci. Am.
Berns, M. W., and Salet, C. (1972) Laser microbeams for partial cell irradiation, Int. Rev. Cytol.
Berns, M. W., Olson, R. S., and Rounds, D. E. (1969a) In vitro
production of chromosomal lesions using an argon laser microbeam, Nature (London)
Berns, M. W., Rounds, D. E., and Olson, R. S. (1969b) Effects of laser microirradiation on chromosomes, Exp. Cell Res.
Berns, M. W., Ohnuki, Y., Rounds, D. E., and Olson, R. S. (1970a) Modification of nucleolar expression following laser microirradiation of chromosomes, Exp. Cell Res.
Berns, M. W., Gamaleja, N., Duffy, C., Olson, R., and Rounds, D. E. (1970b) Argon laser microirradiation of mitochondria in ray myocardial cells in tissue culture, J. Cell Physiol.
Berns, M. W., Cheng, W. K., Floyd, A. D., and Ohnuki, Y. (1971) Chromosome lesions produced with an argon laser microbeam without dye sensitization, Science
Berns, M. W., Rattner, J. B., Brenner, S., and Meredith, S. (1977) The role of the centriolar region in animal cell mitosis: A laser microbeam study, J. Cell Biol.
Berns, M. W., Chong, L. K., Hammer-Wilson, M., Miller, K., and Siemens, A. (1979) Genetic microsurgery by laser: Establishment of a clonal population of rat kangaroo cells (PTK2
) with a directed deficiency in a chromosomal nucleolar organizer, Chromosoma
Bessis, M., Gires, F., and Nomarski, G. (1962) Irradiation des organites cellulaires a l’aide d’un laser a rubis, C. R. Acad. Sci.
Brenner, S. L., Liaw, L.-H., and Berns, M. W. (1980) Laser microirradiation of kinetochores in mitotic PTK2
cells: Chromatid separation and micronucleus formation, Cell Biophys.
Brinkley, B. R., and Pepper, D. (1980) Tubulin nucleation and assembly in mitotic cells: Evidence for nucleic acids in kinetochores and centrosomes, Cell Motil.
Cremer, C., Cremer, T., Zorn, C., and Zimmer, J. (1978) The influence of the distribution of photolesions on the induction of chromosome shattering in Chinese hamster cells by UV-microirradiation and caffeine, Clin. Genet.
Davidson, E. H. (1968) Gene Activity in Early Development
, Academic Press, New York.Google Scholar
Edwards, J. S., Chen, S.-W., and Berns, M. W. (1981) Cereal sensory development following laser microlesions of embryonic apical cells in Acheta domesticus
, J. Neurosci.
Forer, A. (1966) Local reduction of spindle fiber birefringence in living Nephrotoma suturalis
(Loew) spermatocytes induced by ultraviolet microbeam irradiation, J. Cell Biol.
Gould, R. R., and Borisy, G. G. (1977) The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation, J. Cell Biol.
Heidemann, S. R., Sander, G., and Kirschner, M. W. (1977) Evidence for a functional role of RNA in centrioles, Cell
Howard, R. J., and Aist, J. R. (1977) Effects of MBC on hyphal tip organization, growth, and mitosis of Fusarium acriminatrium
, and their antagonism by D2
Kitzes, M., Twiggs, G., and Berns, M. W. (1977) Alteration of membrane electrical activity in rat myocardial cells following selective laser microbeam irradiation. J. Cell Physiol.
Lohs-Schardin, M., Sander, K., Cremer, C., Cremer, T., and Zorn, C. (1979) Localized ultraviolet laser microbeam irradiation of early Drosophila
embryos: Fate maps based on location and frequency of adult defects, Dev. Biol.
McBride, G. M., LaBounty, J., Adams, J., and Berns, M. W. (1974) The totipotency and relationship of seta-bearing cells to thallus development in the green alga Coleochaete scutata.
A laser microbeam study, Dev. Biol.
McNeill, P. A., and Berns, M. W. (1981) Chromosome behavior following laser microirradiation of a single kinetochore in mitotic PTK2
cells, J. Cell Biol.
Moreno, G., Lutz, M., and Bessis, M. (1969) Partial cell irradiation by ultraviolet and visible light. Conventional and laser sources, Int. Rev. Exp. Pathol.
Ohnuki, Y., Olson, R. S., Rounds, D. E., and Berns, M. W. (1972) Laser microbeam irradiation of the juxtanucleolar region of prophase nucleolar chromosomes, Exp. Cell Res.
Peterson, S. P., and Berns, M. W. (1978a) Effect of psoralen and near UV on vertebrate cells in culture: Comparison of laser with standard lamp, Photochem. Photobiol.
Peterson, S. P., and Berns, M. W. (1978b) Chromatin influence on the function and formation of the nuclear envelope shown by laser-induced psoralen photoreaction, J. Cell Sci.
Peterson, S. P., and Berns, M. W. (1978c) Evidence for centriolar region RNA functioning in spindle formation in dividing PTK2
cells, J. Cell Sci.
Rattner, J. B., and Berns, M. W. (1976a) Centriole behavior in early mitosis of rat kangaroo cells (PTK2
Rattner, J. B., and Berns, M. W. (1976b) Distribution of microtubules during centriole separation in rat kangaroo (Potorous)
Rattner, J., Lifsics, J., Meredith, S., and Berns, M. W. (1976) Argon laser microirradiation of mitochondria in rat myocardial cells. VI. Correlation of contractility and ultrastructure, J. Mol. Cell Cardiol.
Salet, C., Moreno, G., and Vinzens, F. A. (1979) A study of beating frequency of a single myocardial cell. III. Laser microirradiation of mitochondria in the presence of KCN or ATP, Exp. Cell Res.
Smith-Sonneborn, J., and Plaut, W. (1967) Evidence for the presence of DNA in the pellicle of Paramecium
, J. Cell Sci.
Strahs, K. R., and Berns, M. W. (1979) Laser microirradiation of stress fibers and intermediate filaments in non-muscle cells from cultured rat heart, Exp. Cell Res.
Strahs, K. R., Burt, J. M., and Berns, M. W. (1978) Contractility changes in cultured cardiac cells following laser microirradiation of myofibrils and the cell surface, Exp. Cell Res.
Tartof, K. D. (1974) Unequal mitotic sister chromatid exchange as the mechanism of ribosomal RNA gene magnification, Proc. Nat. Acad. Sci. U.S.A.
Wilson, C. L., and Aist, J. R. (1967) Mobility of fungal nuclei, Phytopathology
Witt, P. N. (1969) Behavioral consequences of laser lesions in the central nervous system of Araneus diadematus
Cl., Am. Zool.
Zirkle, R. E. (1970) Ultraviolet-microbeam irradiation of newt-cell cytoplasm: Spindle destruction, false anaphase, and delay of true anaphase, Rad. Res.
Zorn, C., Cremer, C., Cremer, T., and Zimmer, J. (1979) Unscheduled DNA synthesis after partial UV irradiation of the cell nucleus. Distribution in interphase and metaphase, Exp. Cell Res.
© Plenum Press, New York 1982