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

Abstract

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...

Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell membrane ruffles

“…contours of the surface are imaged by using the surface scanning mode (secondary electron imaging). Upon mixing the backscattered electron signal with the secondary electron signal, a high-resolution topographical image of cell-surface GLUT4myc labeled with colloidal gold particles is obtained (29). Backscattered electron images reveal all the gold particles as white dots and enable quantitation of the number of goldlabeled epitopes on the cell surface.…”

“…To this end, we used the powerful approach of combined backscatter scanning electron microscopy (29). Myc epitopes on the surface of L6 myotubes were labeled immunologically with anti-myc antibody followed by secondary antibody coupled to colloidal 30-nm-diameter gold particles.…”

Scanning Immunoelectron Microscopy of Hairy Cell Leukemia

“…Immunogold staining permits a direct correlation between cell surface morphology and immunological labeling with many monoclonal antibodies. In addi tion, immunogold SEM coupled with BEI permits to discriminate single gold particles because of their high atomic number from natural surface projections and to evaluate semiquantitatively the intensity of labeling [7,14], Additional advantages of this tech nique are its rapidity, comparable to immunofluores cence and the stability of the labeling which allows the samples to be stored for a long time. Criticism has been raised against phenotypic studies with mono clonal antibodies in HCL since hairy cells tend to bind many antisera nonspecifically [13].…”

“…However, some criticism has still persisted on the reliability of SEM for diagnostic pur poses, mainly because of the potential variability of cell surface morphology. A novel method of scanning immunoelectron microscopy (immuno-SEM), more reproducible and reliable than the previous ones, was recently developed by other authors [7,8,12] as well as in our laboratory [14]. This technique is based on the utilization of monoclonal antibodies as first layer immunoglobulins to which a second layer goat anti-mouse IgG coupled with 20-40 nm colloidal gold par ticles is bound.…”

“…Its second most important feature lies in the fact that the marker, gold particles, is best recognized on the basis of its atomic number contrast by using the backscattered electron imaging (BEI) mode of SEM. In this way, single antigen bound particles can be seen, distinguished from the underlying cell sur face and possibly counted [7,14]. We utilized a panel of monoclonal antibodies with typical reactivity in HCL to test the usefulness of a combined SEM and immunological study of hairy cells.…”