Backscattered electron imaging for immunogold viewing by scanning electron microscope

Immunogold labeling in scanning electron microscopy

“…As the diameter of the gold colloids decreases, however, colloidal gold can hardly be discerned anymore from surface structures or contaminants (dirt particles) of similar size by the structural information alone. It turned out to be much easier to use the backscattered electron (BSE) signal as additional information in order to unambiguously localize colloidal gold (Trejdosiewicz et al 1981;Sieber-Blum et al 1981;Walther et al 1983;Nava et al 1984;DeHarven et al 1984a, b). Hoyer and coworkers (1979), the first to transpose this idea, used X-ray data for this purpose.…”

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.…”

Binding patterns of monoclonal anti‐B, anti‐H and anti‐(Leb+ Y) on erythrocytes, imaged in the scanning electron microscope

“…SEM studies are of special interest, since SEM permits comparison of binding patterns on various parts of morphologically intact cells. Bound antibodies are revealed in SEM by labelling with colloidal gold particles, which are easily visualized by atomic number contrast in the backscatter electron imaging (BEI) mode (12)(13)(14). This mode of imaging has been used in the present study to characterize binding patterns of monoclonal IgM anti-monofucosylated B, anti-H and anti-(Leb + Y) antibodies to erythrocytes by indirect irnmunogold labelling.…”