Microscopica acta最新文献

This communication presents a method for the preparation of azure B-SO2 with trichloracetic acid (TCA) and potassium metabisulphite for in situ demonstration of DNA-aldehyde molecules following acid hydrolysis of tissue sections. The shelf-life of such a dye-reagent is slightly more than that of the control, prepared with N HCl and potassium metabisulphite. The slightly increased shelf-life of the experimental dye-reagent has been considered to be due to a somewhat higher pH as compared with that of the control. The in situ absorption characteristics of nuclei stained for DNA-aldehyde molecules with either an aqueous solution of azure B or with TCA-azure B-SO2 show peak-absorption at 600 nm in both cases. This phenomenon has been interpreted as due to the fact that azure B does not contain any primary amino group in its molecules and, therefore, the mode of binding of DNA-aldehydes with this dye is different from that with dyes that contain primary amino group. The implications of some of the findings have been discussed.

A simple method for quantitative measurements of staining and fluorescence intensities in microscopic preparation is described. The procedure is based on the use of the automatic exposure capability of the Zeiss Photomicroscope, in which the measuring value on the scale of the exposure control device after an arbitrary time permits the assessment of the light intensity on the point photosensor. The method has been checked through the study of chromatin staining and fluorescence by dyes and fluorochromes which are utilized in nucleic acids cytochemistry. The correpondence between these measurements and those obtained by using other methods is briefly discussed.

This communication presents informations on the use of tris-buffer along with N HCl and potassium metabisulphite for the preparation of thionine-SO2 in staining DNA-aldehyde molecules of acid hydrolysed mammalian liver sections. It has been found that thionine, containing tris-buffer, N HCl and potassium metabisulphite, stains DNA-aldehyde molecules with better result than is possible with the control dye-SO2 reagent that does not contain this buffer. The absorption spectra of nuclei stained with this dye-reagent prepared with tris-buffer have also been presented. Further, it has been found that nuclei stained with the freshly prepared dye-SO2 reagent is bluish-violet, whereas those stained with an old dye-reagent is sky blue in colour. The reason for the slightly enhanced nuclear colouration with the experimental dye-reagent over the control has been considered to be due to slightly increased pH in the former as compared with that of the latter. The mechanism of staining with thionine-SO2 has been considered to be of Feulgen type.

Substitution of ethyl alcohol with liquid carbon dioxide, without use of amyl acetate during critical point drying was investigated. Replacement of ethanol was determined by testing for ethanol residue using the Nicotinamide Adenine Dinucleotide method. Usefulness of the method was ascertained.

Nuclei isolated from rat liver can be separated in non-parenchymal diploid, parenchymal diploid and tetraploid nuclei. These nuclei are studied in their chromatin structure using a Barr and Stroud GN5 integrating densitometer. The measuring instrument has the possibility to read an integrated absorbance and as a second value the area of an absorbing object at different levels of extinction. The projection area of Feulgen-stained nuclei were measured at 557 nm at different extinction levels. From the results the conclusion can be drawn that the chromatin structure of parenchymal diploid and tetraploid nuclei is quite the same, however, the chromatin structure of non-parenchymal nuclei is more condensed. This method can be applied for an objective description of the pattern of chromatin density.

An improved device for frequency recording of cyclic movements on the cellular level is described. The device consists of a modified conventional microscope lit by a laser source, an analyzer comprising a fiber optic transmission system, a photodector, an amplifier, a recorder and a thermostatic equipment, including a thermoprobe. In vitro experiments are reported concerning variation in activity as a function of temperature, with respect to the frequency of ciliary beat in rat ring trachea.

The identification of pituitary ACTH and TSH producing cells was performed by histological staining at the isoelectric points (pH levels: 4.6 and 5.2 respectively) of these hormones in cat. Intensity of stained material in the hormone producing cells was determined by microphotometric technique. By the application of hormones influencing the pituitary ACTH and TSH producing relevant changes in pituitary cells could be detected.

The introduction of 1 micron-thick sections from plastic embedded material represents a great technical improvement for the study of tissues under the optical microscope. However, even sections of this thickness appear too thick when observed with oil immersion objectives of high numerical aperture. The extremely shallow depth of field of these lenses allows them to differentiate several focal planes within a one micron thick section. This in turn results in ghost images being formed from out of focus structures, a problem particularly vexing when photomicrography is attempted. To circumvent this difficulty, we reduced the thickness of the sections down to an optimum of 0.4 micron. These thinner sections do require a very energetic stain to give enough contrast to the cellular structures; Stevenel Blue, a stain recently adapted for plastic sections [del Cerro et al., Microsc. Acta 83, (2), 117--121 (1980)] proved to be the most suitable for this purpose of several stains tested. In summary, submicrometer thick sections stained with Stevenel Blue allow to reach the limits of visibility permitted by the best available objectives and effectively merge the realm of optical microscopy with that of low power electron microscopy.

A method for the quantitative evaluation of X-rays is described. The image is decomposed into individual image points by a mechanical scanning procedure, and at each image point the area fraction of a measuring field not covered by silver grains is determined with an image analyzer. This parameter is interpreted as representing a value corresponding to a specific degree of film blackness. The relationship between the measured value and the X-ray absorption is described by standard curves. With the aid of an aluminum scale, the measured value can be expressed directly by the thickness of an aluminum equivalent with a corresponding X-ray absorption. Details about the adjustment of the image analyzer for detecting the silver grains, the resolution of different degrees of X-ray absorption, as well as the computer-controlled scanning procedure are described. An example demonstrates its applicability to analyze the density distribution of bony tissue around the human humero-ulnar joint. The procedure is not limited to the evaluation of X-rays, but is applicable whenever silver grains can be detected in a film layer by an image analyzer.

The separate cellular regions of the reptilian cerebral cortex were studied using Numerical Taxonomy. Three parameters were employed: a) The area occupied by each region at the several levels studied. b) The total area of the cell nuclei present in each level. c) The average are of these nuclei. Numerical Taxonomy resolves the problem by means of a dendrogram which represents the normalised distances, which indicate levels of similarity on absciassas. The elements studies are on the ordinate axis. The dendrogram shows the different levels of similarity existing between each one of the chosen populations. Depending upon the degree of similarity one may deduce the similarities or differences existing between these populations, and also the characteristics of each cellular population throughout the length of its presence in the cerebral cortex and the variations between the regions. These results, in the first place, relate to the four cortical regions: medialis cortex, dorsomedialis cortex, dorsalis cortex, and lateralis cortex, and in the second place, to each one of the regions within the entire telencephalic cortex.