To obtain high resolution backscatter electron (BSE) images in field emission SEM (FESEM), one must consider selection of accelerating voltage, beam current, working distance between the specimen and the backscatter detector (in-lens or below-the-lens position for the specimen), the type of BSE detector, and the type of metal used to coat the specimen to improve conductivity and signal collection [1]. A new generation of below-the-lens FESEM have been tested for BSE imaging on biological samples, but no information exists on whether or not high resolution imaging is possible. Here we report the comparison of detection of a colloidal gold standard (6, 12, 18 nm) by high resolution BSE imaging using Autrata-modified YAG detectors in an in-lens FESEM and in a below-the-lens FESEM.Standards were prepared by mixing colloidal gold particles of 6 nm, 12 nm, and 18 nm. The gold particles were attached via poly-l-lysine to glass chips and coated with <1 nm Pt by ion beam sputtering.
High-resolution immunogold localization of Giardia cyst wall antigens using field emission SEM with secondary and backscatter electron imaging.