Biological volume EM with focused Ga ion beam depends on formation of radiation-resistant Ga-rich layer at block face.

Volume electron microscopy (vEM) enables biologists to visualize nanoscale 3D ultrastructure of entire eukaryotic cells and tissues processed by heavy atom staining and plastic embedding. The vEM technique with the highest resolution is focused ion-beam scanning electron microscopy (FIB-SEM), which provides nearly isotropic (~5-10 nm) spatial resolution at fluences up to 10,000 e/nm. However, it is still not understood how such resolution is achievable, because serial block-face (SBF) SEM, which incorporates an in-situ ultramicrotome instead of a Ga FIB beam, results in radiation-induced collapse of similar specimen blocks at fluences of only ~20 e/nm. Here, we show that FIB-SEM implants a thin concentrated layer of Ga ions, which greatly reduces electron beam-damage, reduces the depth from which backscattered electrons are detected, and prevents specimen charging and collapse. Furthermore, we show that the z-resolution (perpendicular to block-face) in FIB-SEM is substantially higher than predicted by Monte Carlo modeling of the backscattered signal when Ga implantation is not included.