Detection of protein A gold 15 nm marked surface antigens by backscattered electrons

Abstract

Colloidal gold has become an important marker in electron microscopy. Due to its scattering properties colloidal gold is easily detected in TEM. Because of the material dependent signal, preferentially backscattered electrons are used in the SEM. In the present study, we used a YAG (cerium dotted yttrium aluminum garnet) single crystal annular scintillator mounted directly below the objective pole piece. The secondary (SE) and backscattered (BSE) images of protein A gold marked cell surface proteins on two test specimens were compared: Red blood cells with a smooth and yeasts with a highly structured surface. After freeze-drying, the specimens were coated with a layer of carbon (5-10 nm) to enhance conductivity. The specimens were observed in a field emission SEM. In the SE mode a distinction between the gold particles and organic structures was hardly possible; this mode, however, gave good surface information. In the BSE mode, individual gold particles of 10 to 15 nm were easily detected. Thus the combination of the high resolution surface image and the material dependent BSE image provides an accurate localization of the surface antigens.