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 prepared by heavy atom staining and plastic embedding. The highest resolution vEM technique is focused ion-beam scanning electron microscopy (FIB-SEM), which provides nearly isotropic (~5-10 nm) spatial resolution at fluences of > 10,000 e ⁻ /nm ² . However, it is not clear how such high 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 ² . Moreover, the z-resolution (perpendicular to block-face) in FIB-SEM is substantially better than predicted by Monte Carlo modeling of SBF-SEM samples. Here, we show that FIB-SEM implants a thin highly concentrated layer of Ga ⁺ ions at the block surface, which greatly reduces the effect of beam-damage, reduces the depth from which backscattered electrons are detected, and prevents specimen charging and collapse.