High-resolution scanning-densitometry of photographic negatives of human chromosomes. III. Determination of fluorescence emission intensities.

Principles and techniques are discussed for measuring with high topological resolution local emission in fluorescing objects, using photographic negatives. Determination of fluorescence intensities is only possible when an unequivocal relation between the original local fluorescence emission intensities of the object, and the transmittances or densities recorded in the microfluorophotograph is known. This relation is formulated in the theoretical part. From this relation it can be concluded that the recorded intensities can be measured optimally when the optical density values produced by the fluorescence emission fall in the range of the linear portion of the Hurter and Driffield curve. In order to obtain this situation, a uniform low-level pre-exposure of the film emulsion to (white) light is carried out prior to the actual fluorescence emission exposure. This pre-exposure acts to elevate the signal exposure to the linear (steeper) part of the H.D. curve. Inhomogeneity of the excitation beam in the object field, or differences in film emulsion response to the light exposure, will result in erroneous optical densities recorded in the photographic negative. Correction for such artifacts could be obtained by addition of a low concentration of fluorophore to the mounting medium of the microscopic preparation. The overall fluorescent background produced in this way, enabled calibration of local fluorescence intensities in different parts of one fluorophotographic negative, and also of the intensities in different negatives taken from one microscopic preparation. The validity of this approach was checked by comparing data obtained from several photographic negatives of the same quinacrine-stained metaphase, taken with different exposure times to imitate fluctuations in excitation illumination, after conversion of the scanning data into emission intensity values with an alogarithm based on the proposed theoretical relation. In another experiment, fluorescence emission intensities of Feulgen-stained chromosomes which had been measured with a cytofluorometer, were compared with results obtained by conversion of the scanning data measured in the fluorophotographic negatives of the same metaphases. Both types of experiment confirmed the applicability of the procedure described.