High Resolution Backscatter Electron ( Bse ) Imaging of Immunogold with in - Lens and below - the - Lens Field Emission Scanning Electron Microscopes

High resolution backscatter electron (BSE) imaging of colloidal gold with field emission scanning electron microscopy (FESEM) depends upon selection of accelerating voltage, beam current, working distance between specimen and backscatter detector, type of backscatter detector, plus the nature and thickness of the metal coating used to improve conductivity and signal collection. BSE imaging of a gold standard (6 nm, 12 nm, and 18 nm colloidal gold) was investigated with in-lens FESEM using a yttrium-aluminum garnet (YAG) scintillator detector and with below-the-lens FESEM using solid state and scintillator detectors, with the former providing the best imaging. Thin metal coatings (<1 nm) of platinum did not interfere with detection of the gold standard with in-lens FESEM so this thickness of platinum was used to test the BSE imaging of 12 nm gold with either solid state or scintillator detectors in below-the-lens FESEM. Due to the thin metal coatings used, a “charging” phenomenon was seen with secondary electron imaging, but this was negligible with BSE imaging because of their higher energy level. Highest BSE resolution was attained on the gold standard and biological samples with in-lens FESEM, but with below-lens FESEM, the labeling of surface molecules with 12 nm immunogold was successfully detected on coated cells at working distances (WD) of 6-20 mm, although shorter WD provided better quality images.