Abstract
In this paper two examples are presented of the application of laser fluorescent microirradiationto biology. The experiments, performed using a pulsed-laser microfluorometer with high spatial and temporal resolution, concern: (i) a study of the functional state of chromatin and (ii) a study of the fluorescence properties of Hematoporphyrin-derivative in tissue- and culture-cells. (i) The in situ evaluation of the functional state of chromatin has been done in chromosomes and nuclei “in toto” stained with the fluorescent probe Quinacrine Mustard. It has been found that chromatin fractions with different degree of activity, morphologically recognizable at the microscope, present fluorescence decay times markedly different, with a longer decay time for active chromatin. This result has been attributed to the stainability of DNA, which appears to be lower for active chromatin than for the inactive one. A similar result has been obtained in a preliminary experiment on cultures of human lymphocytes, after activation with Phytohemagglutinin. (ii) The fluorescent properties of Hematoporphyrin-derSvative have been studied in single cells, obtained from both normal and tumor tissues and cultures. In agreement with the results obtained with other techniques in tissues, it has been found that the tumor cells examined present an HpD uptake higher than that of the normal cells of the corresponding tissue, and that, within a cell, HpD becomes localized mainly in the cytoplasm. Preliminary results indicate a difference in the fluorescence decay time between stimulated and unstimulated human lymphocytes treated with HpD. Furthermore, it has been found that the fluorescence decay time is different in cells as compared with HpD solution and that the presence of HpD stabilizes cell autofluorescence.
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Literature Cited
A. Andreoni, C. A. Sacchi, and O. Svelto, in: Chemical and Biochemical Applications of Lasers, Vol. 4, C. Bradley Moore (ed.), Academic Press, New York (1979), p. 1.
L. Brand and J. R. Gohlke, Ann. Rev., Biochem.,41, 843 (1972).
N. Böhm, in: Techniques of Biochemical and Biophysical Morphology, Vol. 1, D. Glick and R. Rosenbaum (eds.), Wiley-Interscience, New York (1972), p. 89.
G. Bottiroli, G. Prenna, A. Andreoni, C. A. Sacchi, and O. Svelto, Photochem. Photobiol.,29, 23 (1979).
A. Andreoni, S. Cova, G. Bottiroli, and G. Prenna, Photochem. Photobiol.,29, 951 (1979).
A. Andreoni, A. Longoni, C. A. Sacchi, and O. Svelto, in: The Biochemical Laser, L. Goldman (ed.), Springer-Verlag, Berlin (1981), p. 69.
J. D. Johnson, A. S. Douvas, and J. Bonner, Int. Rev. Cytol., Suppl.,4, 273 (1974).
G. S. Stein, T. C. Spelsberg, and L. J. Kleinsmith, Science,183, 817 (1974).
S. L. McKnight, K. A. Martin, A. L. Beyer, and O. L. Miller, Jr., in: The Cell Nucleus, Vol. 7, H. Busch (ed.), Academic Press, New York-San Francisco-London (1979), p. 97.
M. Durante, P. G. Cionini, S. Avanzi, R. Cremonini, and F. D'Amato, Chromosoma,60, 269 (1977).
D. Serafini Fracassini, N. Bagni, P. G. Cionini, and A. Bennici, Planta (1980).
R. M. Acheson (ed.), Heterocyclic Compounds: Acridines, Vol. IX, Wiley-Interscience, New York (1973); Ritva-Kaisa Selander, Biochem. J.,131, 749 (1973).
G. Bottiroli, P. G. Cionini, F. Docchio, and C. A. Sacchi, in: Lasers in Photomedicine and Photobiology, R. Pratesi and C. A. Sacchi (eds.), Springer-Verlag (1980), p. 175.
J. F. Chiu and L. S. Hnilica, in: Chromatin and Chromosoma Structure, H. J. Li and R. Eckhardt (eds.), Academic Press, New York-San Francisco-London (1977), p. 193.
G. Bottiroli, P. G. Cionini, F. Docchio, and C. A. Sacchi (to be published).
J. D. Braunstein, G. Schwartz, R. A. Good, T. K. Sharpless, and M. R. Melamed, J. Histochem. Cytochem.,27, 474 (1979).
H. Blüthman, Molec. Biol. Rep.,4, 97 (1978).
R. L. Lipson et al., J. Nat. Cancer Inst.,26b, 1 (1961).
H. B. Gregorie Jr. et al., Ann. Surg.,167, 820 (1968).
C. J. Gomer and T. J. Dougherty, Cancer Res.,39, 146 (1979).
A. E. Profio et al., Med. Phys.,6(6), 523 (1979).
T. J. Dougherty et al., Cancer Res.,38, 2628 (1978).
J. Winkelman, J. Nat. Cancer Inst.,27, 1369 (1961).
T. J. Dougherty et al., J. Nat. Cancer Inst.,55, 115 (1975).
F. Docchio et al., Lasers in Surgery and Medicine (to be published).
G. Gomer and D. M. Smith, Photochem. Photobiol.,32, 341 (1980).
G. Jori et al., Tumori,65, 425 (1979).
A. C. Allison et al., Nature,209, 874 (1966).
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Central Electtronica Quantistica e Strumentazione Electtronica, CNR, Istituto di Fisica, Politecnico Milano, Italy. Centro Istochimica, CNR, Istituto di Anatomia Comparata, Università Pavia, Italy. Published in Primenenie Lazerov v Atomnoi, Molekulyarnoi i Yadernoi Fiziki—Trudy II Vsesoyuznoi Shkoly, pp. 121–134, 1981.
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Docchio, F., Ramponi, R., Sacchi, C.A. et al. Laser fluorescent microirradiation: Two examples of application to biology. a. Study of the functional state of chromatin; b. Study of hematoporphyrin derivative (HpD) in cells. J Russ Laser Res 6, 368–376 (1985). https://doi.org/10.1007/BF01125065
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DOI: https://doi.org/10.1007/BF01125065