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Degranulation of chicken heterophil leucocytes during phagocytosis, studied by phase contrast and interference microscopy.

The dynamic aspects of degranulation of chicken heterophil leucocytes during phagocytosis have been studied by phase contrast and interference microscopy. Both standard (8 fps) and high-speed (400 fps) cine-photomicrographic recordings of this process under phase contrast are presented. Lysis of individual granules is usually completed in less than 60 milliseconds. During lysis of each granule a rounded phase dense body is ejected into the phagocytic vacuole. Measurements made by interference microscopy show that there is usually a substantial fall from a protein concentration of c. 100 per cent. w/v for intact granules to a concentration of 16 per cent. w/v for the vacuole resulting from their lysis; this can only be explained by a rapid intake of water into the granule matrix following membrane fusion. The intake of water that accompanies granule lysis causes swelling of the granule matrix, and is thought to explain the mechanism of ejection of the phase dense body. Granule lysis is not dependent on the intake of water, since occasional vacuoles have been observed which showed no fall in protein concentration relative to the intact granules. The membrane around the intact granule effectively excludes the entry of water into the concentrated hygroscopic granule matrix, but once membrane fusion occurs this barrier to the entry of water is usually lost.

Animals

Lysosomal enzyme activities in different types of amniotic fluid cells measured by microchemical methods, combined with interference microscopy.

In primary amniotic fluid cultures, four distinct types of cells were characterized as epithelioid (E I and E II), fibroblast-like (F), And large cells, Small numbers (1-200) of freeze-dried cells were isolated from colonies of each cell type and analyzed for the activity of three lysosomal enzymes: beta-N-acetylglucosaminidase, beta-galactosidase, and alpha-glucosidase. When expressed per cell, the activities for each of the enzymes were not significantly different among the small types of cells (EI, EII, and F). However, 5 to 10-fold higher enzyme activities were found in the large cells. The dry mass of individual large cells, as measured by microinterferometry, was also 5 to 10 times higher than that of the smaller cell types. When expressed per unit of dry mass, the enzyme activities tested, appeared to be independent of the type of amniotic fluid cell. The significance of this observation for the rapid prenatal diagnosis of metabolic diseases is discussed.

Acetylglucosaminidase

Preparation of cells from suspensions for correlative scanning electron and interference microscopy.

Neutrophils from bovine milk and blood platelets from dog plasma were washed in PBS, fixed in GA, dehydrated, suspended in a drop on a formvar-coated slide and immediately critical-point-dried in CO2. After coating with Pt-Pd the specimens were examined in an SEM. The same cells were then examined by interferometry (Int) in a light microscope, and the dry mass was determined. It is shown that this preparation method for both types of microscopes (SEM and Int) appears to give adequate results as far as fine surface structure (SEM-appearance) and dry mass determinations (Int) are concerned. The method has the advantage of a more precise characterization of individual particles, than would have been possible, if both methods of microscopy (SEM and Int) had been employed on the same sample, but on different specimens.

Animals