Reduction of SEM charging artefacts in native cryogenic biological samples

Abstract

Scanning electron microscopy (SEM) of frozen-hydrated biological samples allows imaging of subcellular structures at the mesoscale in a representation of their native state. Combined with focused ion beam milling (FIB), serial FIB/SEM can be used to build a 3-dimensional model of cells and tissues. The correlation of specific regions of interest with cryo-electron microscopy (cryoEM) can additionally enable subsequent high-resolution analysis. However, the use of serial FIB/SEM imaging-based methods is often limited due to charging artefacts arising from insulating areas of cryogenically preserved samples. Here, we demonstrate the use of interleaved scanning to attenuate these artefacts, allowing the observation of biological features that otherwise would be masked or distorted. We apply our method to samples where inherent features were not visible using conventional scanning. These examples include membrane contact sites within mammalian cells, visualisation of the degradation compartment in the algae E. gracilis and observation of a network of membranes within different types of axons in an adult mouse cortex. The proposed alternative scanning method could also be applied to imaging other non-conductive specimens in SEM.