Ultrastructural cytochemical localizations by back-scattered electron imaging of white blood cells.

Soligo, Davide; Harven, E D

doi:10.1177/29.9.6945361

Cited by 15 publications

(5 citation statements)

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“…When such specimens are stained with cytochemical staining methods and observed in the BSE mode, the ezymes in the cell components are seen brightly in a dark view field. In recent years cytochemical studies with SEM have been widely used by means of staining with heavy metal salts and BSE imaging (Abraham & DeNee, 1974;Becker & De Bruyn, 1976;Vogel et al, 1976;DeNee et al, 1977;Soligo & De Harven, 1981). In these studies, however, free cells and thin sections of tissues were used as materials.…”

Section: Discussionmentioning

confidence: 99%

A preparation method for observing intracellular structures by scanning electron microscopy

Tánaka

Mitsushima

1984

Journal of Microscopy

175

122

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SUMMARY In order to observe intracellular structures by scanning electron microscopy, excess cytoplasmic matrix must be removed from the fractured surface of cells. Previously we reported an Osmium‐DMSO‐Osmium method devised for this purpose. This method is very effective in revealing intracellular structures, but requires osmium tetroxide for initial fixation with some consequent disadvantages. In the present study, a revised Osmium‐DMSO‐Osmium method is reported, in which an aldehyde mixture is used as the initial fixative instead of osmium tetroxide. As fixation is carried out by perfusion in this revised method, better preservation of fine structures is achieved than by the original method, especially in the central nervous tissue which tends to suffer from post‐mortem degeneration. Moreover this method can be applied to cytochemical studies of intracellular structures with a scanning electron microscope (SEM). In this study, acid phosphatase of lysosomes is demonstrated in a coloured SEM micrograph.

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Section: Discussionmentioning

confidence: 99%

A preparation method for observing intracellular structures by scanning electron microscopy

Tánaka

Mitsushima

1984

Journal of Microscopy

175

122

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“…However, the cells studied here are more or less spherical in shape and have an average diameter frequently exceeding 5 or 6 pin. We know, from our previous work on the cytochemistry of blood cells viewed in the BEI mode (18), that a 20 kV electron beam does not penetrate cells to such a depth . Current studies, in which different accelerating voltages as well as stereo-pair imaging will be used, are expected to add further support to this statement .…”

Section: Discussionmentioning

confidence: 98%

A novel approach for scanning electron microscopy of colloidal gold-labeled cell surfaces.

Harven¹,

Leung²,

Christensen³

1984

The Journal of Cell Biology

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118

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A method is described for the use of scanning electron microscopy on the surface of gold-labeled cells. It includes the use of 45-or 20-nm colloidal gold marker conjugated with Staphylococcal protein A. The marker is best recognized on the basis of its atomic number contrast by using the backscattered electron imaging mode of the scanning electron microscope . When the backscattered electron signal is mixed with the secondary electron signal, an optimum correlation between the distribution of the labeled sites and the cell surface structures is demonstrated . The method is illustrated by its application to the identification of human circulating granulocytes . Its good resolution, high contrast, and good labeling efficiency offers a promising approach to the specific localization of cell surface antigenic sites labeled with particles of colloidal gold .When reproducible methods for the preparation of cultured cells for scanning electron microscopy (SEM)' were first introduced by Porter et al. (1), it became apparent that the labeling oflarge portions of the cell surface could benefit from the three-dimensional view offered by this instrument. As early as 1972, Lo Buglio et al. (2) ; and two years later, Weller (3), demonstrated that immunoscanning electron microscopy permits the specific correlation of the presence of a given antigen with the surface morphology of well-preserved cells. This was achieved with markers like latex spheres (230 nm in diameter) or haemocyanin molecules (35 x 50 nm in diameter), the sizes ofwhich were compatible with the resolution of the SEM of conductively-coated specimens . These markers were recognized on the basis of their size and/or shape, as it appeared with the SEM in the secondary electron imaging (SEI) mode. Although it is possible to resolve ferritin molecules with higher resolution SEM (4), this marker could not find many practical SEM applications . Results obtained in several laboratories with a variety of markers in the 40-200 nm size range, which includes biological macromolecules (3), viruses (5), and co-polymer microspheres (6), were expertly reviewed by Molday et al . (7) . Markers of such size are unlikely to provide high resolution surface labeling, primarily because steric hindrance phenomena prevent the labeling of many adjacent epitopes (8). In addition, large size markers obliterate too much ofthe underlying surface structure, mak-'Abbreviations used in this paper: BEI, backscattered electron imaging; SEI, secondary electron imaging; SEM, scanning electron microscopy; and TEM, transmission electron microscopy.THE JOURNAL OF CELL BIOLOGY " VOLUME 99 JULY 1984 53-57 C The Rockefeller University Press -0021-9525/84/07/0053/05 $1 .00ing precise topographical relationships difficult to establish.Recently, however, it has been reported that (a) relatively stable conjugates between 5-to 40-nm colloidal gold particles and highly-purified proteins like Staphylococcus aureus protein A or antibodies can be prepared and are readily available (9, 10) ; (b) that c...

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“…Nuclei stained with silver (Gornori 1952) have been shown by numerous investigators with BE1 (Abraham and DeNee 1973. de Harveti et al 1984. Soligo and de Harven 1981: identification and localization of the nucleus may help in the interpretation of cell surface images, and information on nuclear and chromatin morphology can be very useful in the study of transformed or neoplastic cells.…”

Section: B) Visualization Of Intracellular Organelles Stained With Hementioning

confidence: 99%

Biological applications of backscattered electron imaging of scanning electron microscopy

Soligo

Lambertenghi‐Deliliers

1987

Scanning

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Ultrastructural cytochemical localizations by back-scattered electron imaging of white blood cells.

Cited by 15 publications

References 0 publications

A preparation method for observing intracellular structures by scanning electron microscopy

A preparation method for observing intracellular structures by scanning electron microscopy

A novel approach for scanning electron microscopy of colloidal gold-labeled cell surfaces.

Biological applications of backscattered electron imaging of scanning electron microscopy

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