Absorption cytophotometry: Evaluation of three methods for the determination of DNA in feulgen stained nuclei

Summary Three methods for the evaluation of the relative amount of DNA per nucleus by absorption cytophotometry are compared. A combination of the two-wavelength method (Patau, 1952; Ornstein, 1952), the one-wavelength two-area method (Garcia, 1965) and the determination of transmission through nuclear plugs, is proposed in order to estimate systematic errors made by absorption cytophotometry.     

Sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells

A method for sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells is described. Feulgen staining is done first and nuclear DNA estimated by absorption cytophotometry. Following this, the slides are stained with AgNO3. The method has been used to study the process of nucleolar fusion in garlic (Allium sativum L.) meristem root tip cells. It was found that during interphase nucleoli rarely fused, thus most fusion must have occurred before the G1 phase of the cell cycle.     

Sequential Estimation of Nuclear DNA and Silver Staining of Nucleoli in Plant Cells

A method for sequential estimation of nuclear DNA and silver staining of nucleoli in plant cells is described. Feulgen staining is done first and nuclear DNA estimated by absorption cytophotometry. Following this, the slides are stained with AgNO₃. The method has been used to study the process of nucleolar fusion in garlic (Allium sativum L.) meristem root tip cells. It was found that during interphase nucleoli rarely fused, thus most fusion must have occurred before the G_I phase of the cell cycle.     

Cytophotometric assays of ribonucleic acids in Ehrlich tumor cells]

The RNA content was measured in individual mouse Ehrlich ascites tumour cells by absorption cytophotometry after gallocyanin chrome alum staining. The cellular RNA content doubles during the preparation for mitosis and is twice as high as in an hyperdiploid line in an hypertetraploid cell lines.     

Scanning Feulgen-deoxyribonucleic acid cytophotometry of Papanicolaou destained preparations.

Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou destained specimens revealed a differential loss in Feulgen reactivity among human buccal and cervical smears, cultured embryonic lung fibroblasts and invasive cervical carcinoma cells. Loss in Feulgen reactivity in Papanicolaou destained fibroblasts and polyploid nuclei of malignant lesions was observed to result in underestimates of relative Feulgen deoxyribonucleic acid and nuclear area values using scanning integrating microdensitometry. Thus, Papanicolaou stained preparations may not be suitable for deoxyribonucleic acid quantification of high ploidy lesions since distributional absorption error is unpredictably influenced by such factors as ploidy level, nuclear size, chromatin dispersion and differential aldehyde loss during destaining. Feulgen deoxyribonucleic acid cytophotometry of Papanicolaou stained preparations can be useful for differentiating benign from malignant lesions if extent of aneuploidy (as reflected in abnormal deoxyribonucleic acid frequency distribution profile) is used as a diagnostic indicator.     

Retrospective DNa analysis of T3/T4 breast carcinoma using cytophotometry and flow cytometry. A comparative study with prognostic evaluation

Tumor cells from 72 patients with advanced breast carcinoma (T3/T4) were analyzed for their DNA content by cytophotometry and flow cytometry (FCM). Both methods were able to subdivide the tumors into groups with different prognoses. Patients with a normal/near-normal DNa content in the tumor had a better prognosis than did those with aneuploid tumors. FCM measurements of DNA content gave a better discrimination for both survival (P = .019) and disease-free survival (P = .059) than did cytophotometry (P = .105 and P = .067, respectively). These results indicate that FCM analysis of paraffin-embedded material is a more accurate method than is cytophotometry for characterizing the DNA content of breast carcinoma cells. The method could be useful in both retrospective studies and in daily diagnostic work.     

A trial of digital television cytophotometry

A possible application of modern TV analyzers of microimages for absorption cytochemical investigation is considered. Photometric properties of the TV system are evaluated and methodical approaches to their improvement are given. Data on TV and scanning cytophotometry are compared when studying DNA contents in cells of different types.     

Standardized thionin-eosin stain in bronchial cytology. A substitute for hematoxylin-eosin Y staining

A standardized thionin-eosinic acid stain was developed as a quick and highly reproducible staining method for bronchial cytology. Bronchial smears and paraffin-embedded sputum samples were stained with thionin-eosin and with the conventional hematoxylin-eosin Y. Spectral absorption characteristics and staining intensity of thionin-eosin-stained cells were investigated by means of cytophotometry. The staining pattern of thionin-eosin is very close to that of the hematoxylin-eosin stain; the contrast between nucleus and cytoplasm is significantly higher for thionin-eosin. Thionin-eosin can be used for "dye-fixation" of cytologic smears and tissue imprints. Blueing and differentiation (as for hematoxylin-eosin) is not required for thionin-eosin; thus, fixation and staining can be performed within two minutes. The spectral absorption characteristics of thionin-eosin allow reliable automated cytophotometric discrimination of cell nuclei and cytoplasm. The standardized thionin-eosin stain is recommended as a substitute for the hematoxylin and eosin stain in bronchial cytology.     

cytophotometric,flow cytofluorometric and morphometric demonstration of the existence of an antigen dependent thymus lymphocyte subpopulation

Summary Using absorption cytophotometry and flow cytofluorometrical DNA and protein estimation of single thymus lymphocytes we were able to establish that after injection of a large dose of antigen (ovalbumin) a subpopulation of lymphocytes arises in the thymus with high protein contents above that of those lymphocytes normally present, however, in small quantities in the thymus. By morphometrical analysis it was established that these lymphocytes are situated in the outermost cortex.     

Mode of action of amphotericin B on chick embryo fibroblasts and on mouse Ehrlich tumour cells: a cytological and cytochemical analysis.

Chick embryo fibroblasts cultivated in vitro and Ehrlich ascites tumor cells (in vivo or in vitro) have been treated with amphotericin B. Cell multiplication is strongly inhibited. Large clear zones appear in the fibroblast nucleoi (phase-contrast and electron-microscope observations). Many treated fibroblasts and tumour cells have a high DNA content (pre-mitotic or polyploid level; measurements by cytophotometry). However, the RNA content (cytophotometry) and the total protein content (cytophotometry and micro-interferometry) are relatively low in the tumour cells. As shown by autoradiography, DNA synthesis is active but RNA synthesis and, in some cases, protein synthesis are inhibited. Due to this unbalanced growth, the cells cannot divide.     

Cytophotometric determination of unscheduled DNA synthesis

We describe a cytophotometric assay for unscheduled DNA synthesis (UDS) in asynchronously growing cells. Monolayer cultures of human HEp-2 and mouse MCa-11 cells were incubated with the carcinogen methyl-methane sulfonate (MMS), as well as with hydroxyurea and (3H)thymidine. Slides were prepared, and the DNA contents and areas of nuclei were measured by absorption cytophotometry. The labeling of the nuclei, determined on the basis of their DNA content to be in G0/G1, was selectively measured after the preparation of autoradiographs. The labeling of the G0/G1 cells increased with increasing doses of MMS. We also found that the increased nuclear labeling after MMS treatment was not due to induction of replicative DNA synthesis or selective destruction of G0/G1 cells. The results of this assay compared favorably with a standard biochemical method for measuring unscheduled DNA synthesis by benzoylated naphthoylated DEAE cellulose chromatography.     

Determination of the heparin content in basophilic granulocytes by cytophotometry

A method for estimating heparin content in basophilic leucocytes by means of cytophotometry of alcian blue--heparin complex is proposed. Its application for measuring heparin amounts in basophils of healthy donors and of patients with allergies is demonstrated.     

Inhibition of pyrimidine incorporation without inhibition of DNA synthesis.

A putative lymphocytic chalone was tested measuring the incorporation of purine and pyrimidine nucleosides and by cytophotometry. The pyrimidine precursors were inhibited but not the purines. Thymidine and deoxycytidine incorporation even performed simultaneously with cytophotometry can be misleading in the analysis of the inhibition of cell division.     

Determining feulgen-DNA of individual chromosomes by fluorescence cytophotometry with incident light

Summary A microfluorometric method for measuring the DNA content of chromosomes is described. The measurements were made with the MPV (microscope photometer with adjustable measuring diaphragm, E. Leitz, Wetzlar) with an incident light fluorescence illuminator of 0,05% Feulgen (pararosaniline) stained chromosomes of the cell line B 14 FAF 28 of the Chinese hamster. The variation coefficient of the measurements ranged between 4 and 11%. The measured values correlate well with DNA measurements quoted in the literature for absorption cytophotometry and length measurements for the same chromosomes.With support from the Deutsche Forschungsgemeinschaft, AZ Mu 258/3.Parts of the results reported here are submitted by Barnim Nitsch to the medical faculty of Munich in fulfilment of the requirements for the Dr. med. degree.We thank Dr. K.-R. Trott, Gesellschaft für Strahlenforschung mbH, Neuherberg, for the original cell cultures of the Chinese hamster; Dr. F. Back, Institut für Hämatologie der GSF, München, for allowing us to use the MPV; Dr. K. Moritz, Zoologisches Institut der Universität München, for carrying out the absorption measurements on the UMSP I.     

Determination of DNA surplus in the nuclei of some cerebellar Purkinje cells with different cytochemical methods

Cytofluorometry and cytophotometry (Feulgen staining) as well as ultraviolet cytophotometry revealed similar histograms of DNA content in squashed nuclei of rat cerebellar Purkinje cells. More than 90% diploid (2c) cells and 1% tetraploid (4c) cells were obtained. DNA content in other Purkinje cells ranged between the 2c and 4c level.     

OFF-PEAK ABSORPTION MEASUREMENTS IN FEULGEN CYTOPHOTOMETRY

The use of off-peak measurements in Feulgen cytophotometry is reported, using intact diploid (x) and tetraploid (y) nuclei of the human anterior pituitary gland as the experimental model studied. Results indicate that the ratio y/x is similar at the 14 wavelengths examined over the range 450 mµ to 650 mµ. The value of this ratio was 1.95, falling slightly under the theoretical ratio of 2.0. It was concluded that off-peak absorption measurements are of value in Feulgen cytophotometry. A discussion of the possible justification of off-peak absorption measurements was presented for the case in which the Beer-Lambert relationship was experimentally determined to be followed at a peak wavelength, for all concentrations under discussion, and the curves preceding and/or following the peaks were straight lines coming from a common point. If the curve best fitting the data is a straight line, it follows that the rate of change of absorbance with respect to a linear wavelength scale is constant. This means that for a given increase, or decrease, in wavelength, the same change in absorbance is obtained. From this it follows that absorbance readings at any wavelength in such a region will be equally valid to those taken at the peak. While the finding of such a linear relationship at several concentrations does not guarantee that it will occur at all other concentrations, it is suggestive. The closer the spectra approximate straight lines, the more valid does the use of off-peak measurements become.     

Further observations on the alleged presence of non-argyrophile argentaffin cells in the human gastro-intestinal tract

Summary The relationship between the argentaffin and argyrophile cells of the human gastro-intestinal tract has been studied by staining paraffin sections first by the Masson-Hamperl method and then by the Bodian method. The investigations confirms the author's earlier observation (Singh 1963) that all argentaffin cells of the human gastro-intestinal tract are also argyrophile and that there is no evidence to support the claim of Hellweg (1952) and of Hamperl (1952) regarding the presence of non-argyrophile argentaffin cells.     

Quantitative estimation of pituitary gonadotropin in juvenile rainbow trout with scanning cytophotometry applied to immunocytochemical preparations

A densitometrical method with computer-controlled scanning cytophotometry has been developed to quantify the amount of gonadotropin (GTH) in immunocytochemically treated paraffin sections of pituitaries of juvenile rainbow trout. Results obtained with this method totally correspond with those established with a radioimmunoassay of pooled pituitaries. Scanningcytophotometry offers the advantage that GTH-content of individual pituitaries of juvenile or small adult fish can be investigated.     

A Cytophotometric Technique for Measuring Photosynthetic Potential of Leaves: Preliminary Research and Development

A novel combined photographic and cytophotometric technique provides information on the physiological state of leaves and a permanent record of each leaf measured in field studies. The method has the ability to determine the distribution of photosynthetic potential within various portions of the leaf and to scale up to the canopy level as well as down to the cellular level. Photographic transparencies are taken in the field, then brought back to the lab to be analyzed cytophotometrically at the investigator's convenience. The image encoded on the film yields an absorption curve that is similar to that of intact leaves with peaks in the blue (450 nm) and red (660 nm) for paper birch. Internal standards of fluorescein and first surface mirrors combined with standardized magnification and illumination (e.g., ring flash) are used to insure precision and accuracy. Two wavelength and plug cytophotometric equations have been modified for use in this technique. Some problems with the two-wavelength method remain, but the usefulness of the plug method for cytophotometry has been expanded through the use of portable leaf area and chlorophyll meters and portable photosynthesis laboratories. Total photosynthetic potential (TPP) is shown to equal leaf area multiplied by the mean optical density of the leaf. With the use of internal standards TPP can be expressed in fluorescein units or adjusted by optical density of the image of the first surface mirror.     

A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.