Synopsis
Bearing in mind the numerous advantages offered by fluorescence measurements, in particular their greater sensitivity as compared with absorption cytophotometry, the possibility of measuring cellular DNA using the conventional Feulgen reaction in cytofluorometry has been studied. By means of a series of direct and indirect comparisons, it is shown that the data obtained by conventional Feulgen reaction cytofluorometry are equivalent to those obtained by integrating microdensitometry, provided that the following experimental conditions are observed:
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(1)
The extinction of the preparations should not exceed 0.1. In general, extinction values lower than 0.1 can be obtained by hydrolysing for the usual times, by using a 0.01% Schiff reagent and 480 nm as the excitation wavelength.
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(2)
The spectral zones subject to inner filter effects are excluded from the measurements, by employing barrier filters RG 630, RG 665 and RG 695, depending on the case.
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(3)
The error due to photo-decomposition is eliminated or reduced by operational devices (which are described).
General aspects of fluorescence quantitation, whether or not the inner filter effect is present, are discussed. Certain special characteristics of an original fully-digitalized electronic instrumentation for high-sensitivity cytofluorometry and cytospectrofluorometry by multi-channel scaling and single photon detection, are also illustrated.
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Invited paper at the ‘Conference on Quantitative Fluorescence Techniques as Applied in Cell Biology’, organized by the Battelle Institute, Seattle, 27–31 March 1972.
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Prenna, G., Leiva, S. & Mazzini, G. Quantitation of DNA by cytofluorometry of the conventional Feulgen reaction. Histochem J 6, 467–489 (1974). https://doi.org/10.1007/BF01003265
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DOI: https://doi.org/10.1007/BF01003265