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Laser Microirradiation and Computer Video Optical Microscopy in Cell Analysis

  • Michael W. Berns
  • Robert J. Walter

Abstract

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.

Keywords

Acridine Orange Nucleolar Organizer Focus Laser Beam Functional Kinetochore Focus Laser Spot 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. 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. 5:5598.CrossRefGoogle Scholar
  2. 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 1:291–302.CrossRefGoogle Scholar
  3. 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 1:275–289.CrossRefGoogle Scholar
  4. Berns, M. W. (1972) Partial cell irradiation with a tunable organic dye laser, Nature (London) 240:483.CrossRefGoogle Scholar
  5. Berns, M. W. (1974a) Biological Microirradiation (Biological Techniques Series), Prentice-Hall, New York.Google Scholar
  6. Berns, M. W. (1974b) Directed chromosome loss by laser microirradiation, Science 186:700.CrossRefGoogle Scholar
  7. 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
  8. Berns, M. W. (1976) A possible two-photon effect in vitro using a focused laser beam, Biophys. J. 16:973.CrossRefGoogle Scholar
  9. Berns, M. W., and Cheng, W. K. (1971) Are chromosome secondary constrictions nucleolar organizers? A re-evaluation using a laser microbeam, Exp. Cell Res. 69:185.CrossRefGoogle Scholar
  10. Berns, M. W., and Floyd, A. D. (1971) Chromosome microdissection by laser: A functional cytochemical analysis, Exp. Cell Res. 67:305.CrossRefGoogle Scholar
  11. Berns, M. W., and Richardson, S. M. (1977) Continuation of mitosis after selective laser microbeam destruction of the centriolar region, J. Cell Biol. 75:977.CrossRefGoogle Scholar
  12. Berns, M. W., and Rounds, D. E. (1970) Cell surgery by laser, Sci. Am. 22:98.CrossRefGoogle Scholar
  13. Berns, M. W., and Salet, C. (1972) Laser microbeams for partial cell irradiation, Int. Rev. Cytol. 33:131.CrossRefGoogle Scholar
  14. Berns, M. W., Olson, R. S., and Rounds, D. E. (1969a) In vitro production of chromosomal lesions using an argon laser microbeam, Nature (London) 221:74.CrossRefGoogle Scholar
  15. Berns, M. W., Rounds, D. E., and Olson, R. S. (1969b) Effects of laser microirradiation on chromosomes, Exp. Cell Res. 56:292.CrossRefGoogle Scholar
  16. 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. 60:133.CrossRefGoogle Scholar
  17. 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. 76:207.CrossRefGoogle Scholar
  18. 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 171:903.CrossRefGoogle Scholar
  19. 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. 72:351.CrossRefGoogle Scholar
  20. 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 73:1.CrossRefGoogle Scholar
  21. Bessis, M., Gires, F., and Nomarski, G. (1962) Irradiation des organites cellulaires a l’aide d’un laser a rubis, C. R. Acad. Sci. 225:1010.Google Scholar
  22. 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. 2:139.Google Scholar
  23. Brinkley, B. R., and Pepper, D. (1980) Tubulin nucleation and assembly in mitotic cells: Evidence for nucleic acids in kinetochores and centrosomes, Cell Motil. 1:1.Google Scholar
  24. 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. 14:286.CrossRefGoogle Scholar
  25. Davidson, E. H. (1968) Gene Activity in Early Development, Academic Press, New York.Google Scholar
  26. 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. 1:250–258.Google Scholar
  27. Forer, A. (1966) Local reduction of spindle fiber birefringence in living Nephrotoma suturalis (Loew) spermatocytes induced by ultraviolet microbeam irradiation, J. Cell Biol. 25:95.CrossRefGoogle Scholar
  28. Gould, R. R., and Borisy, G. G. (1977) The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation, J. Cell Biol. 73:601.CrossRefGoogle Scholar
  29. Heidemann, S. R., Sander, G., and Kirschner, M. W. (1977) Evidence for a functional role of RNA in centrioles, Cell 10:337.CrossRefGoogle Scholar
  30. 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 D2O, Protoplasma 92:195.CrossRefGoogle Scholar
  31. 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. 93:99.CrossRefGoogle Scholar
  32. 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. 68:533.CrossRefGoogle Scholar
  33. 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. 37:90.CrossRefGoogle Scholar
  34. McNeill, P. A., and Berns, M. W. (1981) Chromosome behavior following laser microirradiation of a single kinetochore in mitotic PTK2 cells, J. Cell Biol. 88:543–553.CrossRefGoogle Scholar
  35. Moreno, G., Lutz, M., and Bessis, M. (1969) Partial cell irradiation by ultraviolet and visible light. Conventional and laser sources, Int. Rev. Exp. Pathol. 7:99.Google Scholar
  36. 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. 71:132.CrossRefGoogle Scholar
  37. 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. 27:367.CrossRefGoogle Scholar
  38. 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. 32:197.Google Scholar
  39. Peterson, S. P., and Berns, M. W. (1978c) Evidence for centriolar region RNA functioning in spindle formation in dividing PTK2 cells, J. Cell Sci. 34:289.Google Scholar
  40. Rattner, J. B., and Berns, M. W. (1976a) Centriole behavior in early mitosis of rat kangaroo cells (PTK2), Chromosoma 54:387.CrossRefGoogle Scholar
  41. Rattner, J. B., and Berns, M. W. (1976b) Distribution of microtubules during centriole separation in rat kangaroo (Potorous) cells, Cytobios 15:37.Google Scholar
  42. 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. 8:239.CrossRefGoogle Scholar
  43. 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. 120:25.CrossRefGoogle Scholar
  44. Smith-Sonneborn, J., and Plaut, W. (1967) Evidence for the presence of DNA in the pellicle of Paramecium, J. Cell Sci. 2:225.Google Scholar
  45. 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. 119:31.CrossRefGoogle Scholar
  46. 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. 113:75.CrossRefGoogle Scholar
  47. Tartof, K. D. (1974) Unequal mitotic sister chromatid exchange as the mechanism of ribosomal RNA gene magnification, Proc. Nat. Acad. Sci. U.S.A. 71:1272.CrossRefGoogle Scholar
  48. Wilson, C. L., and Aist, J. R. (1967) Mobility of fungal nuclei, Phytopathology 57:769.Google Scholar
  49. Witt, P. N. (1969) Behavioral consequences of laser lesions in the central nervous system of Araneus diadematus Cl., Am. Zool. 9:121.Google Scholar
  50. Zirkle, R. E. (1970) Ultraviolet-microbeam irradiation of newt-cell cytoplasm: Spindle destruction, false anaphase, and delay of true anaphase, Rad. Res. 41:516.CrossRefGoogle Scholar
  51. 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. 124:111.CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1982

Authors and Affiliations

  • Michael W. Berns
    • 1
  • Robert J. Walter
    • 1
  1. 1.Department of Developmental and Cell BiologyUniversity of California, IrvineIrvineUSA

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