High-resolution immunogold labeling of cell surfaces: The hypothetical homodimeric nature of the cd5 receptor

Abstract

Molecules exposed on cell surfaces and labeled with colloidal gold markers can be optimally demonstrated using the backscattered electron imaging (BEI) mode of the scanning electron microscope (SEM). Steric hindrance, however, limits labeling efficiency, making it necessary to use gold markers of small size for labeling at the molecular level. Using a JEOL 840 SEM equipped with a lanthanum hexaboride (LaB6) cathode, 13 nm gold particles were demonstrated. This, however, seems to represent the limit of the resolution of this type of instrument in the BEI mode. Fortunately, it has been demonstrated by Walther and Muller that 5 nm gold particles can be seen in the BEI mode, using field emission SEM.We have confirmed this observation, using the JEOL 890 field emission SEM and a solid state backscattered electron detector. Human peripheral blood lymphocytes prefixed with 0.1% glutaraldehyde, incubated with the murine monoclonal antibody LEU-1 (CD5), were labeled with a goat anti-murine IgG adsorbed on 5 nm gold particles (GAM-G5, Janssen Pharmaceutica, Beerse, Belgium) according to previously described procedures.