Scanning electron microscopy of tobacco mosaic virus labeled lymphocyte surface antigens

Abstract

The study of surface antigens by immunoelectron microscopy has been hampered by the fact that thin sections of cells provide only a view of the cell perimeter in an essentially 2 dimensional fashion. Although the reconstruction of the entire cell from serial sections has been accomplished, it remains too exacting a technique and will find only exceptional application. Carbon platinum replicas allow the inspection of larger surface areas and therefore are better suited for studying the distribution of antigens. But since only relatively smooth surfaces will yield stable replicas, cells with large numbers of microvilli are not amenable to this technique. Despite its limited resolution, scanning electron microscopy (SEM) is the method of choice because it can provide a view of almost half of the surface of a cell close to its natural configuration, particularly after critical point or freeze drying. Immunological labeling methods have not yet been routinely applied to SEM although both latex spheres and hemocyanin have been used with some success. The optimal visual marker should possess the following properties: be of a distinctive shape, chemically stable, and have per se a low binding affinity for cell surfaces. Tobacco mosaic virus (TMV), a marker which is familiar in transmission electron microscopy, meets these demands; it has rod like shape and defined dimensions (15 X 300 nm) and in addition it can easily be distinguished from surface microvilli. As the hybrid antibody technique (9) is also applicable to TMV, it was attempted to combine such immunological labeling with SEM. Evidence is presented that surface antigens can be visualized by SEM, using the TMV marker in conjunction with the hybrid antibody technique.