Ga + focused ion beam scanning electron microscopy (Ga + FIB-SEM) enables cell biologists to visualize nanoscale 3D ultrastructure of entire eukaryotic cells and tissues prepared by chemical fixation, heavy atom staining, and epoxy-resin embedding. Monte Carlo simulations of electron scattering in such samples predict that the spatial resolution perpendicular to the block-face should be limited to ∼20 nm yet, experimentally, Ga + FIB-SEM provides nearly isotropic (∼5 to 10 nm) spatial resolution. Furthermore, experience with serial block face (SBF)-SEM, which uses an in-situ diamond knife instead of an ion beam, shows that the specimen collapses at electron fluences > 20 e - /nm 2 limiting the signal-to-noise in the 3D images, whereas specimen blocks analyzed in the FIB-SEM can withstand fluences of ∼10,000 e - /nm 2 at a beam energy of 1.5 keV. By imaging 3D ultrastructure of embedded neurons in the Ga + FIB-SEM, we have shown that a previously unknown but highly concentrated surface layer of Ga + ions is implanted within a 20-nm thick layer at the block face, which greatly reduces the effect of beam-damage and reduces the depth from which backscattered electrons are detected. We show that individual 40-nm diameter synaptic vesicles are resolved as hollow membrane shells in the FIB-SEM. We are currently assessing the achievable resolution in 3D images containing other supramolecular structures.
Leapman et al. (Sun,) studied this question.