High-resolution scanning-densitometry of photographic negatives of human chromosomes

Abstract

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.

Determination of nuclear DNA of five Eucoccidian parasites, Isospora (Toxoplasma) gondii, Sarcocystis cruzi, Eimeria tenella, E. acervulina and Plasmodium berghei, with special reference to gamontogenesis and meiosis in I. (T.) gondii

“…The photomultiplier was an EMI 9558 with an S20 cathode (EMI electronics Ltd, Hayes, Middlesex, U.K.), operated on a stabilized high-voltage supply, type MFLK BN S/N 396 (Knott Elektronik AG, Munich, F.R.G. ; Van der Ploeg, Vossepoel, Bosman & Van Duijn (19776)). Photometric readings were performed using EPI-illumination from an HBO 100 high-pressure mercury arc (Osram GmbH, Berlin, F.R.G.)…”

Cytochemical studies on nuclear DNA of four eucoccidian parasites,Isospora (Toxoplasma) gondii, Eimeria tenella, Sarcocystis cruziandPlasmodium berghei

“…The excess of Schiff reagent was removed by washing in sulphurous acid (3 x 10 min) and in Mcllvaine buffer (citric acid/disodium hydrogen phosphate, pH 56) for 45 min as described by Duijndam & Van Duijn (1973. After dehydration via a graded alcohol-xylene series, the slides were mounted in Fluoromount (Gurr, High Wycombe, Bucks, U.K.) to which Gargille oil nfj = 1460 (Cargille Lab., Cedar Grove, N.J., USA) was added (15:1 v/v), resulting in a mixture with a refractive index of about 154 (Van der Ploeg, Van Duijn & Ploem, 1974). To control for unspecific binding of the Feulgen stain, non-hydrolysed preparations were stained and investigated in an identical manner.…”

“…(This film is no longer available and was replaced by Kodak technical pan film 2415 during the present experiments.) After pre-exposure of the photographic emulsion to homogeneous light, or a colour reversal film (Agfachrome 50L) was used (Van der Ploeg et al 1974;Van der Ploeg, Vossepoel, Bosman & Van Duijn, 1977).…”