Important parameters for the detection of small colloidal immunogold particles (10 nm) on cell surfaces include the use of field emission SEM (FESEM) and a need for a conductive metal coating to reduce charging, so as to facilitate backscatter electron (BSE) imaging for unambiguous identification by atomic number contrast. Recently Heinzmann et al (1) have reported the use of below-lens FESEM for detection of 10 nm colloidal gold at working distances of 6-13 mm, but achieved success only on uncoated cells as coating with a 1 nm layer of platinum appeared to mask the gold signal. To improve specimen conductivity and signal collection, three metals (Cr, Pt, W) have been used to generate thin coatings either by planar magnetron sputtering for cryoSEM or ion-beam sputtering for routine chemically-fixed, critical point dried samples (2,3)

Three below-lens FESEM (Hitachi S-4500, JEOL 630land 63401, and LEO 982) were tested by using a sample of human neutrophils which had been chemically fixed, then immunostained for the surface antigen CD43 with 10 nm colloidal gold, and prepared by critical point drying.