Staining of Muscle Tissue for Quantitative Studies and Automated Morphometry

Seventy-six staining tests were carried out on paraffin sections of human and animal muscle to find a suitable staining method for quantitative morphometry of muscle fibers. The results were evaluated under the light microscope, on black and white photomicrographs and on an image analysing computer, the Quantimet 720. A brilliant scarlet-phosphotungstic acid-tartrazine method is described and recommended for automated morphometry after additional testing on 140 sections of developing human muscle using the Quantimet 720.     

Change of chromatin morphology during the cell cycle detected by means of automated image analysis

Mouse embryo fibroblasts growing asynchronously in vitro stained with Feulgen method and their nuclear chromatin was analysed by means of the image analysing computer Quantimet 720D. Cells with 2C, 3C and 4C content of DNA were considered as being in G₁, middle S and G₂ phase of cell cycle, respectively. It was found that the projected area of nuclei increases during the cell cycle and that the mean optical density of chromatin increases from G₁ through S to G₂ phase. The curves showing the areas of chromatin at different optical density thresholds are different for cells in G₁, S and G₂ phase. The results demonstrate cyclic changes in chromatin morphology in the interphase nuclei during the cell cycle.     

Staining of muscle tissue for quantitative studies and automated morphometry.

Seventy-six staining tests were carried out of paraffin sections of human and animal muscle to find a suitable staining method for quantitative morphometry of muscle fibers. The results were evaluated under the light microscope, on black and white photomicrographs and on an image analysing computer, the Quantimet 720.A brilliant scarlet-phosphotungstic acid-tartrazine method is described and recommended for automated morphometry after additional testing on 140 sections of developing human muscle using the Quantiment 720.     

Methods of measuring swimming speed of spermatozoa.

Three basic approaches for determining the mean swimming speed of a suspension of microorganisms were compared, using bull and ram spermatozoa. Number fluctuation counting was performed automatically on a Quantimet 720 image analysing computer, the mean speed being obtained using 'probability after' statistics. The other two approaches were photomicrographic: number flux counting was performed on single photomicrographs; on the same photomicrographs, the mean speed was estimated from measurement of 'whole' and 'half' track lengths. These results were compared with each other and with the Quantimet results. The 'probability after' method was also compared, on additional samples, with cine-photomicrographic tracking. The mean speeds predicted by the 'probability after' method compared favourably with the other methods (range 68 mum/sec to 162 mum/sec). The results also suggested that, on single photomicrographs, measurement of 'half' track lengths or number flux counting were generally preferable to measurement of whole track lengths.     

Effect of food-deprivation on rat pancreatic islet cells

Somatostatin, insulin and glucagon concentrations in rat pancreas were measured following various intervals of food-deprivation. Tissue concentrations, as measured by radioimmunoassay, were correlated with A-, B-, and D-cell number and size using a scanning integrating image analyzer (Quantimet 720). Alterations in total islet hormone content were not correlated to changes in size or distribution of cells. This implies that changes in tissue content reflect changes in turnover of peptides rather than changes in cell size or number.     

Immunocytochemical localisation of prolactin and growth hormone in the perinatal sheep pituitary

Summary The peroxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestational age of the fetus may influence the size range of prolactin secretory granules.     

A quantitative analysis of photometric data from aerial photographs for vegetation survey

This paper describes the application of quantitative density analysis to black and white aerial photographs for vegetation survey using a Quantimet 720 image analyser. The photometric data are analysed using both a supervised and an unsupervised classification strategy. Floristic data collected from an independent ground survey, are used to categorise those vegetation classes of interest against which the photometric data classifications are assessed. The preliminary results obtained suggest that broad classes of vegetation types may be distinguished automatically from their grey scale distribution patterns.     

The measurement of histochemically-stained NADP-dependent isocitrate dehydrogenase in the sebaceous glands of hairless hamster.

The effect of substrate, co-factor and Nitroblue Tetrazolium concentration on the production of formazan by the action of NADP-dependent isocitrate dehydrogenase was studied in the sebaceous glands of the hairless hamster. The measurement (in average optical density units per unit area) of formazan in histochemically stained skin sections was carried out by television scanning microdensitometry (Quantimet 720D). Having established optimal conditions for this enzyme, a second study was carried out to determine the effect of different power objectives and wide-band wavelength light instead of white light on the average optical density per unit area, recorded by the instrument, of the formazan produced in a defined number of sebaceous glands in a single skin section. It was found that there was no difference in the average optical density per unit area recorded by the instrument at different power objectives and a peak value of average optical density per unit area could be obtained using a K4 or a K5 Balzer filter (550-650 nm).     

Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary

Summary Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.     

A new method for automatic metaphase finding adaptable to different chromosome preparations

A FORTRAN computer program, running on a Digital PDP 11-34 minicomputer, has been developed for use in conjunction with a Cambridge Quantimet 720 image analyzer for the investigation of metaphase preparations in routine cytogenetics. During a short initiation phase the program is adapted to the type of metaphase being analyzed. The program is fast and its performance is good, even at low microscopic magnifications. It has other uses in biology for all investigations and characterizations of small distinct elements widely spread within a preparation (e.g., autoradiography, bacteriology).     

Comparison of two techniques for the quantitative measurement of acid phosphatase in normal and regenerating epithelium of the female hairless hamster

Synopsis Two methods have been employed for measuring acid phosphatase activity in normal and regenerating epithelium of the female hairless hamster. The first method entails histochemical preparation followed by television scanning microdensitometric measurements using a Quantimet 720D. The second method involves incubation of freeze-dried epidermis in a suitable substrate medium followed by measurement of the fluorescence of the released product by a spectrophotofluorometer. The results were compared against each other by performing best-fit polynomial regression line analysis to the data produced by both methods. A correlation coefficient of 0.99 was obtained when interpolated data from the two best-fit curves were compared with each other. This indicates that the histochemical technique and the subsequent measurement using television scanning microdensitometry is quantitative and can, therefore, be employed in quantitative histochemistry.     

Sizes and numbers of nuclei in the cortex of thymus glands of red-billed weavers Quelea quelea

The cortex of the thymus glands of embryos, chicks, juveniles, fledglings and adults from several colonies of Quelea quelea were studied using an image analyser (Quantimet 720) to determine cell populations and nuclear sizes. Just prior to hatching the lobes showed a high level of mitosis and consisted of predominantly small lymphocytes. The larger glands of chicks and juveniles had higher cell populations; pyknotic cells and erythrocytes occurred free in the cortex. The lobes of adults were more variable but in general mitosis occurred in enlarging glands of adults from colonies with eggs; most lobes contained pyknotic cells but not in such high numbers as in lobes from chicks and fledglings. Erythrocytes were common, occurring in large numbers in the cortex in some birds. The factors affecting the interpretation of these data are discussed in detail.     

Toluidine blue binding capacity of heterochromatin and euchromatin of Triatoma injestans Klug

Summary Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO₃, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

Quantitation of the immunocytochemical assay for estrogen receptor protein (ER-ICA) in human breast cancer by television imaging

A Quantimet 720D Image Analysis System has been programmed for light microscopic evaluation of the nuclear estrogen receptor distribution in frozen sections of human breast cancer stained by the peroxidase-antiperoxidase method using monoclonal antibodies to estrogen receptor protein (ER). This method provides precise criteria for distinguishing ER-positive and -negative cells and a sensitive and reproducible means for densitometric quantification of the staining patterns. Although imaging sequence and graphic analysis are automated by computer programs, light pen interaction provides supervision of feature selection. Imaging of the immunocytochemical assay (ER-ICA) in 50 patients revealed marked heterogeneity of nuclear estrogen receptor concentration varying over a nearly 100 fold concentration range. Various ER concentration patterns were evident: (I) distributions with a single peak (CV = 5%) present at various concentration levels; (II) bimodal distributions, revealing co-existent ER-positive and ER-negative subpopulations; (III) multimodal distributions with a number of resolvable concentration peaks; and (IV) highly skewed distributions with or without discernible peaks, frequently extending over the entire concentration range. Statistical methods of de-convolution were applied to determine the frequency and ER concentration characteristics of component subpopulations in the mosaic cases and for resolving the proportion of ER-positive and -negative cells. An approach for evaluating nuclear ER content in conjunction with ER concentration patterns in individual patients revealed whether spread in the ER concentration distribution resulted from differences in nuclear ER content or from variability in nuclear volume distribution.     

Micro-biomechanics vs macro-biomechanics in cortical bone. A micromechanical investigation of femurs deformed by bending

Previous studies have shown that longitudinal and transverse lamellae in compact bone signal the presence of tensile and compressive forces, respectively. On this basis an investigation has been carried out to ascertain the distribution of lamellae in femoral shafts that have been deformed by bending. To do this, a series of undecalcified plane-parallel sections 100 microns thick were prepared, and the proportion of transversely oriented collagen and crystallites in the lamellae was measured, using circularly polarized light as an illuminating source and a Quantimet 720 image-analyzing computer. It has been concluded that the distribution of the two types of lamellae is in accordance with the need to compensate for the deformity produced by the bending of the bones, and that an obvious relationship exists between the macroscopic features of the femurs and their modified microscopic structures.     

Effect of point substitutions of Asp-714 and Asp-720 residues on the structure and function of the H+-ATPase of the yeast plasma membrane

Membrane-spanning M5 and M6 segments, which play a role in the formation of cation transport sites in H⁺-, Ca²⁺-, K⁺-, Na⁺-, and other P2-ATPases, are connected by a short extracytoplasmic loop. In the yeast plasma membrane H⁺-ATPase, which belongs to a family of P2-ATPases, the loop is connected to M5 and M6 through the Asp-714 and Asp-720 residues. In this work, the effect of point amino acid replacements of Asp-714 and Asp-720 by Ala, Val, Asn, and Glu residues on the function of the enzyme was studied. The D714A point mutant possessed activities similar to those of the wild-type enzyme, whereas the replacement of Asp-714 by other amino acid residues disrupted biogenesis and led to a loss of activity. All mutants with substitution of Asp-720 were expressed and possessed relatively high activity. The D720V mutant displayed significantly reduced expression level, activity, H⁺ transport and its coupling to ATP hydrolysis. Thus, substitutions of Asp-714, except for the D714N mutant, led to significant defects in biogenesis and/or function of the enzyme. The results indicate the important role for the Asp-714 residue in biogenesis, structure stability, and enzyme function.     

Treatment with lithium salts reduces ethanol dehydration shrinkage of glutaraldehyde fixed tissue

Changes in the area of glutaraldehyde fixed 15 day p.c. mouse embryo limbs were recorded using a Quantimet 720 image analysing computer attached to a light microscope: during a period of treatment with an isotonic salt solution (mostly halides of the alkali or alkaline earth metals); a subsequent wash with distilled water; and dehydration through a 30, 50, 70, 80, 90, and 100% ethanol series. Pretreatment with NaCl, KCl, RbCl had no significant effect. Treatment with LiCl, LiNO3, LiF (0.03 M), CsF and CsCl caused an increase (relative to Na, K or Rb treated samples) in the specimen volume during dehydration, which persisted in 100% ethanol. Li treated samples showed the largest post-critical-point-drying (CPD) volumes, followed by Cs treated tissue. Pretreatment with Be, Mg, Ca, Sr and Ba chlorides caused shrinkage and the 100% ethanol and post-CPD volumes of these samples were all lower than those treated with the monovalent cation containing salts.     

Nitrite réductase des Saccharomyces (groupe Torulaspora) et des Debaryomyces. implications systématiques

Intracellular nitrite reductase was found in some species of Saccharomyces (Torulaspora group of Van der Walt 1970) and all Debaryomyces (excepted D. castelli and D. coudertii. The taxonomical relationship between the 2 groupes are discussed.

     

Identification of two Robertsonian translocations with a Giemsa banding technique

Summary Using a trypsin banding technique a D/D and a G/G translocation were identified as D-13/14 and G-21/22.

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Zusammenfassung Mit Hilfe einer Trypsin-Bandierungstechnik wurde eine D/D-und eine G/G-Translokation als D-13/14 und G-21/22 identifiziert.