Abstract
Fluorescence analysis provides a sensitive, selective, nondegradative approach to the measurement of nucleic acids in intact cells. Fluorophores of high quantum yield can be detected in very low concentrations, and, by the analysis of fluorescence quantities such as intensity, spectra, polarization, and decay time, it is possible to obtain significant information about the environment of the fluorophore. The position and interaction of the fluorophore with the intact structures within a cell can be determined. Usually this is done after fixation of the cell, because this allows access through the cell membrane for the probe and other reagents. There are a number of fluorophores that complex selectively with nucleic acids, but few that complex only with DNA or RNA. Selectivity can be improved by enzymatic digestion of RNA or DNA before application of the probe. Although fixed cells offer these technical advantages, there are many reasons for probing cells that retain full integrity, metabolic capacity, and viability. Recent reports indicate that this is becoming possible, and it is hoped that this review will help to stimulate further activity in this promising field.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Arndt-Jovin, D., and Jovin, T., 1976, Analysis by computer-controlled cell sorter of Friend virus-transformed cells in different stages of differentiation, Haematol. Blutransfus. 19:137–149.
Arndt-Jovin, D., and Jovin, T., 1977, Analysis and sorting of living cells according to deoxyribonucleic acid content, J. Histochem. Cytochem. 25:585–589.
Arndt-Jovin, D., and Jovin, T., 1978, Automated cell sorting with flow systems, Ann. Rev. Biophys. Bioeng. 7:527–558.
Berns, M., 1977, Biological, photochemical, and spectroscopic applications of lasers, Photochem. Photobiol. Rev. 2:1–38.
Burns, V., 1972, Location and molecular characteristics of fluorescent complexes of ethidium bromide in the cell, Exp. Cell Res. 75:200–206.
Burns, V., 1977, Studies with a fluorescent vital probe for DNA in mammalian cells, Exp. Cell Res. 107:459–462.
Cowden, R., and Curtis, S., 1976, Some quantitative aspects of acridine orange fluorescence in unfixed, sucrose-isolated mammalian nuclei, Histochem. J. 8:45–49.
Crissman, H., and Steinkamp, J., 1973, Rapid, simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations, J. Cell Biol. 59:766–771.
Crissman, H., and Tobey, R., 1974, Cell-cycle analysis in 20 minutes, Science 184:1297–1298.
Crissman, H., Oka, M., and Steinkamp, J., 1976, Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells, J. Histochem. Cytochem. 24:64–71.
Darzynkiewicz, Z., Traganos, F., Sharpless, T., Friend, C., and Melamed, M., 1976a, Nuclear chromatin changes during erythroid differentiation of Friend-virus-induced leukemic cells, Exp. Cell. Res. 99:301–309.
Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M., 1976b, Lymphocyte stimulation: A rapid multiparameter analysis, Proc. Nat. Acad. Sci. USA 73:2881–2884.
Darzynkiewicz, Z., Traganos, F., Arlin, Z., Sharpless, T., and Melamed, M., 1976c, Cytofluorometric studies on conformation of nucleic acids in situ, J. Histochem. Cytochem. 24:49–58.
Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M., 1977a, Cell cycle related changes in nuclear chromatin of stimulated lymphocytes as measured by flow cytometry, Cancer Res. 37:4635–4640.
Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M., 1977b, Interphase and metaphase chromatin: Different stainability of DNA with acridine orange after treatment at low pH, Exp. Cell. Res. 110:201–214.
Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M., 1977c, Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation, J. Cell Biol. 73:128–138.
Darzynkiewicz, Z., Traganos, F., Sharpless, T., and Melamed, M., 1977d, Recognition of cells in mitosis by flow cytofluorometry, J. Histochem. Cytochem. 25:875–887.
Darzykiewicz, Z., Andreef, M., Traganos, F., Sharpless, T., and Melamed, M., 1978, Discrimination of cycling and noncycling lymphocytes by BudR-suppressed acridine orange fluorescence in flow cytometric system, Exp. Cell Res. 115:31–36.
Fowlkes, B., Herman, C., and Cassady, M., 1976, Flow microfluorometric system for screening gynecologic specimens using propidium iodidefluorescein isothiocyanate, J. Histochem. Cytochem. 14:322–329.
Golden, J., West, S., Echols, C., and Shingleton, H., 1976, Quantitative fluorescence spectrophotometry of acridine orange stained unfixed cells, J. Histochem. Cytochem. 24:315–321.
Gray, J., Carrano, A., Steinmetz, L., Van Dilla, M., Moore, D., Mayall, B., and Mendelsohn, M., 1975, Chromosome measurement and sorting by flow systems, Proc. Nat. Acad. Sci. USA 72:1231–1234.
Hawkes, S., and Bartholomew, J., 1977, Quantitative determination of transformed cells in a mixed population by simultaneous fluorescence analysis of cell surface and DNA in individual cells, Proc. Nat. Acad. Sci. USA 74:1626–1630.
Heinen, E., Bassleer, R., and CalbErg-Bacq, C., 1976, Comparison of the effects of ethidium bromide-deoxyribonucleic acid complex in fibroblasts cultivated in vitro, Beitr. Path. Bd. 159:207–218.
Hilwig, I., and Gropp, A., 1975, pH-depEndent fluorescence of DNA and RNA in cytologic staining with 33258 Hoechst, Exp. Cell Res. 91:457–461.
Huang, C., and Baserga, R., 1976, Circular dichroism studies of ethidium bromide binding to the isolated nucleolus, Nucleic Acids Res. 3:1857–1873.
Ito, T., 1973, Diffusion of acridine molecules to a genetic site in living cells, Biochim. Biophys. Acta 329:140–146.
Ivanovic, V., Geacintov, N., and Weinstein, D., 1976, Cellular binding of benzopyrene to DNA characterized by low-temperature fluorescence, Biochem. Biophys. Res. Commun. 70:1172–1179.
Jensen, R., 1977, Chromomycin Aa as a fluorescent probe for flow cytometry of human gynecologic samples, J. Histochem. Cytochem. 25:573–579.
Jensen, R., Langlois, R., and Mayall, B., 1977, Strategies for choosing a deoxyribonucleic acid stain for flow cytometry of metaphase chromosomes, J. Histochem. Cytochem. 25:954–964.
Latt, S., 1973, Microfluorometric detection of DNA synthesis in human chromosomes, Proc. Nat. Acad. Sci. USA 70:3395–3399.
Latt, S., 1977, Fluorometric detection of deoxyribonucleic acid synthesis; Possibilities for interfacing bromodeoxyuridine dye techniques with flow fluorometry, J. Histochem. Cytochem. 25:913–926.
Latt, S., and Stetten, G., 1976, Spectral studies on 33258 Hoechst and related bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid synthesis, J. Histochem. Cytochem. 24:24–33.
Latt, S., and Wohlleb, J., 1975, Optical studies of the interaction of 33258 Hoechst with DNA, chromatin, and metaphase chromosomes, Chromosoma 52:297–316.
Latt, S., Stetten, G., Juergens, L., Willard, H., and Scher, C., 1975, Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence, J. Histochem. Cytochem. 23:493–505.
Latt, S., George, Y., and Gray, J., 1977, Flow cytometric analysis of bromodeoxyuridine substituted cells stained with 33258 Hoechst, J. Histochem. Cytochem. 25:927–934.
Lawrence, J., and Daune, M., 1976, Ethidium bromide as a probe of conformational heterogeneity of DNA in chromatin, Biochemistry 15:3301–3307.
Melamed, M., Mullaney, P., and Mendelsohn, M., 1978, Flow Cytometry and Sorting, Wiley, New York.
Nicolini, C., Kendall, F., Desaive, C., Baserga, R., Clarkson, B., and Fried, J., 1976, Physical-chemical characterization of living cells by laser-flow microfluorometry, Cancer Treat. Rep. 60:1819–1827.
Parry, G., and Hawkes, S., 1978, Detection of an early surface change during oncogenic transformation, Proc. Nat. Acad. Sci. USA 75:3703–3707.
Schwarzacher, H., Gropp, A., and Ruzicka, F., 1976, Fine structure of 33258 H-treated chromosomes, Hum. Genet. 33:259–262.
Slavik, M., and Carter, S., 1975, Chromomycin Aa, mithramycin, and olivomycin: antitumor antibiotics of related structure, Adv. Pharmacol. Chemother. 12:1–30.
Steinkamp, J., Hansen, K., and Crissman, H., 1976, Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells, J. Histochem. Cytochem. 24:292–297.
Stockert, J., 1974, Ethidium bromide fluorescence in living and fixed cells, Naturwissenschaften 61:363–364.
Stubblefield, E., 1975, Analysis of the replication pattern of Chinese hamster chromosomes using bromodeoxyuridine suppression of 33258 Hoechst fluorescence, Chromosoma 53:209–221.
Swartzendruber, D., 1977, Microfluorometric analysis of cellular DNA following incorporation of bromodeoxyuridine, J. Cell. Physiol. 90:445–454.
Traganos, F., Darzynkiewicz, Z., Sharpless, T., and Melamed, M., 1976, cytofluorometric studies on conformation of nucleic acids in situ, J. Histochem. Cytochem. 24:40–48.
Traganos, F., Darzynkiewicz, Z., Sharpless, T., and Melamed, M., 1977, Simultaneous staining of ribonucleic acid and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system, J. Histochem. Cytochem. 25:46–56.
Van de Sande, J., Lin, C., and Jorgenson, K., 1977, Reverse banding on chromosomes produced by a guanosine-cytosine-specific DNA-binding antibiotic: olivomycin, Science 195:400–402.
Van Dilla, M., Trujillo, T., Mullaney, P., and Coulter, J., 1969, Cell microfluorometry: A method for rapid fluorescence measurement, Science 163:1213–1214.
Varesio, L., Capuccinelli, P., and Forni, G., 1975, Inhibition of replication of virus-transformed fibroblasts by antibodies to RNA, Cancer Res. 35:3558–3565.
Watson, J., and Chambers, S., 1977, Fluorescence discrimination between diploid cells on their RNA content: A possible distinction between clonogenic and nonclonogenic cells, Br. J. Cancer 36:592–600.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1980 Plenum Press, New York
About this chapter
Cite this chapter
Burns, V.W. (1980). Fluorescent Probes in the Study of Nucleic Acids and Chromatin in Living Cells. In: Smith, K.C. (eds) Photochemical and Photobiological Reviews. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-3641-9_3
Download citation
DOI: https://doi.org/10.1007/978-1-4684-3641-9_3
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4684-3643-3
Online ISBN: 978-1-4684-3641-9
eBook Packages: Springer Book Archive