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...