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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

Comparison of buccal and nasal epithelial cells using a new cell development index and quantitative interference microscopy.

Cell area, cell dry mass, and three summary indices for tissue maturation were obtained for human buccal and nasal cells viewed in smears (five donors). The means for cell area and dry mass in the buccal smears were 3816 X 10(-8) cm2 and 2294 pg (1 pg=1 X 10(-12) g), respectively. The correlation coefficient for means of cell area and dry mass was r=0.03. The means for the three maturation indices used to characterize the cells viewed in buccal smears were: Cell Development Index (CDI), 486; Maturation Index (MI) 50.6; and Karyopyknotic Index (KPI), 0.028. The correlation coefficients for means were: CDI vs. MI, r=0.88; CDI vs. KPI, r=0.71; and MI vs. KPI, r=0.73. In the buccal smears, the cell types found most frequently were intermediate with round or oval nuclei, which altogether averaged about 90% of the total smear composition, while superficial cells (pyknotic ghost, and anucleate) comprised less than four per cent. In nasal smears, the means for cell area and dry mass were 867 X 10(-8) cm2 and 623 pg, respectively. The correlation coefficient for means of cell area and dry mass was r=0.74. The means for the maturation indices were: CDI, 816; MI, 91.2; and KPI, 0.79. The correlation coefficients for means were CDI vs. MI, r=0.93; CDI vs. KPI, r=0.97; and MI vs. KPI, r=0.85. In nasal smears, the anucleate cell type was found most frequently (about 75%) while intermediate cells with round or oval nuclei comprised approximately 13% of the total smear composition. When donor means for cell area and dry mass of buccal cells were combined for study, they were highly correlated (r=0.88). Similarly, when the maturation indices for buccal and nasal cells were combined for study, they were highly correlated: CDI vs. MI, r=0.99; CDI vs. KPI, r=0.99; and MI vs. KPI, r=0.99. Of the three indices, we believe that the CDI is the index best suited for use in studies of cell development because it is based on an inferred sequence of cellular and nuclear changes occurring during the maturation of epithelial cells. It provides a more detailed description of the cells observed in the smears. It also correlates highly in KPI and MI used in clinical and research efforts.

Cell Nucleus

The surface structure of ageing human articular cartilage: a study by reflected light interference microscopy (RLIM).

The surface contours of the weight-bearing area of the left lateral femoral condyle have been examined in vitro in 26 cadavers by reflected light interference microscopy over the age range 0--47 years. The presence of 4 orders of surface irregularity was confirmed. In addition, measurement of the depth, diameter and frequency of the tertiary order of surface hollows showed that while none of these variables was sex-associated, age-related changes in the variables were present. Multiple regression analysis showed that these changes could be considered in terms of periods of maturation (0--21 years) and ageing (22--50 years) of the cartilage. Focal changes in the surface were observed with advancing age. These changes appeared to be regressive and possibly represent an early stage in cartilage fibrillation.

Adolescent

[Selective contrast enhancement of microscopical specimen by optical procedures for automatized quantitative image analysis (author's transl)].

One of the most important problems in automatic image analysis is the discrimination of features by, a certain range of grey levels. To obtain as many as possible different grey levels sufficient contrast is achieved mainly by specific staining. Alternative and supplementary methods are some micrscopical methods, not routinely used. Interference microscopy, interference reflexion microscopy and microfluorimetry are discussed in detail. These optical procedures enhance the contrast of specimen specifically without the necessity for the application of specimen specifically without the necessity for the application of sophisticated staining methods. In interference contrast, tissues can be separately detected by grey level discrimination due to varying concentration of dry mass; this is shown for a cornifying part of fish skin (breeding tubercle of Rutilus rutilus L.). Furthermore, very small amounts of dry matter can be determined with high precision, as demonstrated for a single tissue culture cell (XTH-cell). Automatisation of image analysis provides a unique opportunity for routine application of interferometric measurements. The principles of the procedures are outlined. By interference reflexion microscopy cellular attachment areas to a glass surface are visualized, providing a powerful tool in cellular diagnosis based on grey level discrimination (darkest parts correspond to zones of closest contact to the substratum). A fast migrating lymphocyte and stationary endothelial cells have been chosen for demonstration. Various histochemical problems can be solved elegantly by fluorescence methods, e.g. mitochondria in living cells are specifically stained by a fluorochrome (DASPMI) and the distribution of fluorescence intensity can be followed within the mitochondrial population of a cell. Fluorescence was recorded from fotographic negatives taken with a fluorescence microscope. Additionally a short comment is given on the application of polarisation microscopy for feature detection.

Animals

T-mycoplasmas: a study of the morphology, ultrastructure and mode of division of some human strains.

The morphology of 10 strains of T-mycoplasma was studied in wet preparations of viable cells by darkfield, phase-contrast and interference microscopy, and in fixed preparations by various techniques of electron microscopy. Mycoplasma-like artefacts in the horse-serum component of the medium were eliminated by filtration. All 10 strains were similar. Individual cells were spherical, 0-25-1-0 mum in size, with a bounding trilaminar membrane, 10 nm thick and containing 7-5-12-5-nm particles, and a layer of pilus-like projections, 5-8 nm long, on the outer surface. A possible capsular matrix was observed only by the pseudoreplica technique. The cells contained 12-15-nm ribosomes, nuclear fibroids 7-5-9 nm wide, and vacuoles. During replication, the cell elongated slightly and the ribsomes migrated to the ends of the cell leaving a ribosome-free area into which the bounding membrane invaginated to form a bud. The bud eventually separated by completion of the process of invagination; a cross-septum did not form. Usually only a single bud developed but sometimes two appeared simultaneously.

Cell Membrane

A method of permanently mounting biological tissue cleared in Herr's four-and-a-half clearing fluid.

A technique which should be generally applicable for preparing permanent mounts of tissue cleared in Herr's four-and-a-half clearing fluid is described. This technique involves transferring plant or animal tissues through a series of solutions consisting of Pienarr's fixative, Herr's clearing fluid, chloral hydrate, acetone and finally polyester resin for mounting. Material prepared using this method is exceptionally transparent and well preserved, and is suitable for either phase contrast or Nomarski interference microscopy.

Chloral Hydrate

[The application of interference contrast microscopy in biology (author's transl)].

Like phase contrast, Nomarski interference contrast microscopy can be used to examine unstained specimens in biology and medicine. The properties of both contrast enhancement techniques are illustrated by various examples. The phase contrast method is especially suited for thin specimens with small differences in refractive index, whereas the interference contrast method supplies good results even of thick specimens. Interference contrast is a valuable supplement to the phase contrast method, and expands the application of microscopy in biology

Animals

Sputum screening by Nomarski interference contrast microscopy.

Gram-stained smears of specimens submitted for sputum cultures were compared with direct wet mounts examined by Nomarski interference contrast microscopy (NIM) for enumeration of squamous epithelial cells (EPC) and leukocytes (WBC). The results obtained by the two methods were comparable, but specimens were more rapidly screened and cell types were more readily differentiated by NIM. Specimens submitted for sputum culture over a 3-month period were examined for EPC and WBC by NIM. Twenty-two percent of the specimens had greater than 25 EPC/field or a predominance of EPC (class I), 30% had greater than 25 EPC and greater than 25 WBC/field (class II), and 48% had greater than 25 WBC/field or a predominance of WBC (class III). The clinical relevance of the culture results was determined by reviewing the records of patients whose specimens were included in the study. Class I specimens provided only 30% clinically relevant culture results. Specimens in class II provided useful culture results in 63% of the patients, and 96% of those in class III provided clinically relevant information. The results confirm the value of sputum screening and demonstrate that NIM provides a rapid, simple, and accurate method for sputum screening.

Diagnosis, Differential

Observations of the microcirculatory bed in rat mesocecum using differential interference constrast microscopy in vivo and electron microscopy.

The microvascular bed of the rat mesocecum has been examined in vivo using differential interference (Nomarski) optics and subsequently by electron microscopy. The preferential channel, from terminal arteriole to collecting venule, has been examined. In the terminal arteriolar segment the endothelial layer is covered by a continuous layer of smooth muscle cells which, in turn, are surrounded by adventitia. In the metarteriolar segment the periendothelial cells still resemble smooth muscle cells but the tunica media is discontinuous. In the distal segment periendothelial cells are more scattered and have the appearance of pericytes. There appears to be a continuous transition of the periendothelial cell layer from terminal arteriole to distal segment. Nerve endings were seeen in both the terminal arteriolar and metarteriolar segments. During contraction smooth muscle cells, oriented circumferentially, shorten and thicken. Endothelial cells appear anchored by myoendothelial junctions. Endothelial cells have filaments which show increased banding during vasoconstriction, suggesting that such cells may contract. Capillary offshoots leave the preferential channel, usually at right angles. Smooth muscle cells are oriented to form a sphincter and there are many myoendothelial junctions at the branch point. Within a short distance the capillary branch loses its periendothelial coat.

Animals

Encephalitis in suckling rats induced with rat cytomegalovirus.

Encephalitis has been induced in suckling rats by intracerebral inoculation of rat cytomegalovirus. This agent has been carried through five animal passages without changes in virulence. Principal sites of viral attack were the meninges and ependyma with limited parenchymal invasion from these sites. Characteristic features of cytomegalovirus disease observed included cytomegaly, formation of nuclear and cytoplasmic inclusions, and polykaryocytosis. Fundamental tinctorial and optical differences were noted between nuclear and cytoplasmic inclusions, the latter being characterized by their phloxinophilia which contrasts with the hematoxylinophilia of nuclear inclusions. Differential interference microscopy improved visualization and photography of cytoplasmic inclusions. Hydrocephalus developed in a few of the animals and was studied in late phases of the disease.

Animals

[Early stages of myocardial infarct studied by opitcal methods].

The hearts of 45 dogs were investigated at times from 10 minutes to 24 hours following ligation of the anterior interventricular branch of the left coronary artery. The material was fixed in formalin and embedded in parafin. It was established that at early stages of experimental myocardial infarction in the zone of eschemia there took place eschemic zone various foms of contactural damages, lumpy decmposition, relaxation and sarcoplasmic matrix. By the polarizing microscope method in the peripheral area of the eshemic zone various forms of contractural damages, lumpy decomposition, relaxation and desaggregation of myofibrills, were noted. Phaso-contrast and interference microscopy investigations revealed in the damaged cells an induration of the sarcoplasmic matrix due to an increase in the dry matter concentration. The studies conducted with the help of polarizing, phaso-contrast, and interference microscopy methods showed that by using these methods it is possible at early periods to reveal the zone of myocardial infarction on stained and unstained preparations prepared by conventional methods.

Animals

Interference reflection microscopy of adhesion of Amoeba proteus.

A simple method of observing the adhesive behaviour of large cells by means of interference reflection microscope is described, and some observations of monopodial Amoeba proteus are presented. Amoebae may contact with the substratum at any point along the long axis of the cells. Points of contact are usually few, small and temporary. Frequently the cell surface within the contact points oscillates, changing the separation distance from the substratum.

Amoeba