Microscopica acta最新文献

This paper presents methods for specific staining of nuclei with aqueous solutions of celestin blue B and gallocyanine in tissue sections from which RNA has been extracted selectively with concentrated phosphoric acid at 5 degrees C for 20 min or by hydrolysis in 6 N HCl at 28 degrees C for 15 min. It has been found that pH of the freshly prepared celestin blue B dye solution is 3.0 and that of an aqueous solution of gallocyanine is 2.8. These pHs can be lowered to 1.5 with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But with concentrated sulphuric or nitric acid and at this pH staining of the nuclei is possible. But if the pHs are lowered with concentrated hydrochloric or phosphoric acid, effective use of these dyes is not possible. It has been suggested that some dispersion of the two dyes takes place with concentrated sulphuric or nitric acid which are used to lower the pH. Staining of the nuclei is also possible with an aqueous solution of celestin blue B at pH 3.0 but the same is not possible with gallocyanine at pH 2.8. The absorption spectra of nuclei stained with an aqueous solution of celestin blue B at pH 1.5 and 3.0 are fairly identical, the peak of maximum absorption being at 620 nm. Those of nuclei stained with an aqueous solution of gallocyanine reveal irregular peaks. Possible implications of these findings have been discussed.

A method for automated quantitative analysis of single label microautoradiographs applied to DNA repair synthesis is proposed. It uses image analysis of the microscopic field by a Leitz TAS and allows an automated selection of the nuclei to be analysed. The use of a grey level gradient algorithm provides a relatively large independence from external variables such as staining intensity, nuclei density in the field, stability of illumination etc. Random choice of the microscopic field by the computer directed stage displacement and automatic focusing ensures a high degree of objectivity and independence from human intervention. The coefficient of correlation between the automatic and the visual grain count is 0.97 in the range of visually countable grain densities, while the precision of measurement is about 10%. The speed of measurement is satisfactory (40 to 60 s for the analyse of a microscopic field) taking in account that only occasional supervision is needed.

Investigations concerning cell densities and projection areas in layered tissues, which use the procedures published by Haug, are burdened with problems due to the division of the layers and the dependence on the size of the evaluation field. In order to find a more exact division, we used a modified microscopic field. Experiments show that a circular field has no advantage as compared with a quadratic field. Therefore, the circular field cannot be recommended.

Three-dimensional structures of the core of herpes virus of turkeys were examined by computerized reconstruction from electron micrographs. There was a core consisting of six small particles, and a core consisting of a toroid surrounding a cylindrical mass. It is suggested that the cylindrical mass developed from six small nuclear particles.

This paper reports on the use of some Schiff-type dye-reagents prepared with oxalic acid and sodium thiosulphate. Some of the dyes, such as azure A, azure B, toluidine blue O, thionine, brilliant cresyl blue, and methylene violet were also prepared with oxalic acid and sodium thiosulphate but fortified with disodium hydrogen phosphate. It has been found that all these dye-reagents, barring neutral red-SO2, when used on acid-hydrolysed mammalian tissue sections under UV rays yield far better staining of the DNA-aldehyde molecules than is possible in controls stained under normal laboratory conditions. Sections stained with any of the dye-reagents under UV rays do withstand treatment in SO2 water or acid water without showing any leaching of the dye from the nuclei. Aqueous solutions of all these dyes can also be used to stain DNA-aldehyde molecules of acid-hydrolysed tissue sections. But sections stained with aqueous solutions of the azures, safranine, phenosafranine, and methylene violet when treated with SO2 water reveal considerable leaching of the dye from the nuclei. However, leaching does not occur when these dyes are used as SO2-containing dye-reagents under UV rays. It has, therefore, been concluded that in the case of staining with the azures, safranine, phenosafranine, and methylene violet, containing SO2, the reaction under UV rays is of Feulgen-type. In the case of staining with toluidine blue-SO2, thionine-SO2, and brilliant cresyl blue-SO2 under UV rays, the mechanism of staining is also of Feulgen-type but it is due to liberation of the dye molecules from their more or less colourless states that react with the DNA-aldehyde molecules producing nuclear colouration as though these dyes are in aqueous solution. This interpretation is based on the fact that sections from which RNA has been extracted selectively with cold concentrated phosphoric acid when treated with these dye-reagents under UV rays also stain DNA-phosphate groups. The increased staining intensity of the nuclei produced with the majority of the dye-reagents under UV rays as compared with control sections stained under usual laboratory conditions has been considered to be due to electronic excitation of the dye molecules thus facilitating binding between more molecules of DNA-aldehyde and of dye. Possible significance of these findings has been discussed.

Cells from smears of the normal human squamous epithelium of the gingiva, fixed and stained for protein using the tetrazonium method optimized by Nöhammer and calibrated by Nöhammer et al., were investigated. The extinctions of both the total cells and of the rectangular areas circumscribing the nuclei, were measured microspectrometrically. Altogether 417 cells from 6 healthy persons of both sexes were investigated. 9 distinct subgroups of cells were found showing an exact linear correlation between nuclear and total cell extinctions. In the graph of both the nuclear and the total cell extinctions the 9 subgroups can be seen as 9 distinct linear groups of points, defined exactly by their regression lines. Thus, every squamous epithelial cell within the smear can be typed definitely and objectively in respect to its membership of one of these 9 linear groups of points. The obviously definite, legitimate connection between the extinctions of the total cells and of their nuclei affords a glimpse into the processes of cellular differentiation and allows the definition of the so-called stem cell in terms of protein content of the total cell and of the nucleus.

Development and amplification of a Quantimet 720 image analyser are reported in combination with a microprocessing unit. The system controlling the measuring procedure with telecommunication for evaluation in a large computer is presented in detail. The new cost-saving units supplementing the analyser render possible: i) reduction of measuring times, ii) reduction of possible errors of measurement, and iii) optimal rational evaluation of data in a large computer system.

To obtain a reliable and fast quantification in low cell density tissues, we have developed a standardized procedure based on image analysis. The successive steps of quantification, starting with the necessary histological procedure and ending with the desired quantitative parameters, are described. Excepting the histological procedure, we have attempted to automate all the subsequent steps of the experiment, especially the acquisition of the data by an image analyser TAS (Leitz), and the storage and the analysis of these data by a PDP 11-34 computer. The final parameters calculated by such a method are the total number of cells and the cell density within the organ under study, either for all the cells, or for different cellular categories. This procedure has been applied to the quantitative study of cerebellar cells of an adult Staggerer mutant mouse and the results obtained are presented.

Medial gastrocnemius (MG) and soleus (S) histochemical fiber type profiles were determined in female albino rabbits (n = 5). The fiber composition of the central S was 93.5 +/- 12.7 S.D.% slow-twitch oxidative (STO), and 6.5 +/- 12.7% fast-twitch oxidative glycolytic (FTOG). No fast-twitch glycolytic (FTG) fibers were found in any of the S muscles. The S FTOG fiber percentage varied from a minimum of 0% to a maximum of 29% in different rabbits. The composition of the central MG was 22.1 +/- 2.0% STO, 28.8 +/- 4.6% FTOG, 43.9 +- 3.5% FTG, and 5.2 +/- 2.5% unidentified fibers. In the MG the STO fiber percentage increased anteriorly, while the FTG fiber percentage increased posteriorly. Two methods were employed to determine fiber diameters. Using the mean of orthogonal diameters method, we found central S diameters to be 74.6 +/- 5.1 S.D. micrometers for STO, ad 67.7 +/- 7.9 micrometers for FTOG fibers. Diameters for the central MG were 62.1 +/- 7.2 micrometers for STO, 66.1 +/- 9.4 micrometers for FTOG, 95.9 +/- 9.3 micrometers for FTG, and 83.3 +/- 9.1 micrometers for unidentified fibers. Fiber diameters calculated by the mean of orthogonal diameters method were for the S, 1.16 +/- 0.01, and for the MG, 1.21 +/- 0.01, times larger than those obtained using the "smallest diameter" (maximum minor diameter) technique. The relationship between these measurement parameters varied in the different muscles due to fiber shape variation. In the central S, STO fibers occupied 94.6%, and FTOG, 5.4% of the cross sectional area, using areas derived from the mean of orthogonal diameters measurements. In the central MG, the relative areas were 13.2% for STO, 19.6% for FTOG, 61.6% for FTG, and 5.6% for unidentified fibers. Relative area calculations were comparable using both of the measurement methods. Therefore, relative area comparisons tend to discount variations introduced by different measurement techniques.

This communication presents informations on the staining of DNA with basic dyes, such as ethyl violet, Janus green, a new red dye obtained from Janus green, and Giemsa, belonging to different chemical groups. It has been found that DNA of tissue sections from which RNA has been extracted selectively with cold concentrated phosphoric acid can be stained with aqueous solutions of these dyes. Further, DNA of tissue sections from which RNA has been extracted and the sections then hydrolysed in 6 N HCl at 28 degrees C for 15 min can also be stained with aqueous solutions of ethyl violet, Janus green, and Giemsa. Moreover, tissue sections that have been hydrolysed in 6 N HCl and then stained with aqueous solutions of these dyes, including the new red dye obtained from Janus green, reveal well-stained nuclei. The absorption spectra of nuclei stained with aqueous solutions of ethyl violet, Janus green and Giemsa, following the above stated three procedures of staining have been presented. Absorption data of nuclei stained with the new red dye after Feulgen hydrolysis of tissue sections as well as those of nuclei in tissue sections from which RNA has been extracted have also been presented herein. The implications of these findings have been discussed.