IMAGING OF IMMUNOLABELED MEMBRANE-RECEPTORS IN UNCOATED SEM SPECIMENS

Epidermal growth factor receptors (EGFR) were labeled with 10 nm immun ogold and examined on uncoated specimens of A431 human epidermoid carc inoma cells. A field emission gun and a high-sensitivity YAG ring dete ctor were used to demonstrate the affinity labeling simultaneously in the secondary-electron (SE) and backscattered-electron (BSE) modes wit h a low accelerating voltage (V(o)). At V(o) = 2 kV, the SE and BSE si gnals were too weak to identify all markers, while at V(o) = 3-7 kV la beling was observed unambiguously in both the SE and BSE modes with sm aller and higher working distances. Increasing the V(o) to above 7 kV sometimes provokes instability of the specimens. A V(o) of greater-tha n-or-equal-to 10 kV produces charging artifacts in the SE image, but p ermits a BSE image of the gold markers providing additional topographi c information. In conclusion, immunogold labeling can be used with goo d results for uncoated specimens.