A protocol for cryogenic volumetric imaging using serial plasma FIB/SEM.

ABSTRACT

Cryogenic volumetric imaging using serial plasma focused ion beam scanning electron microscopy (serial pFIB/SEM) is a new and exciting correlative volume electron microscopy (vEM) technique. It enables visualization of un-stained, cryogenically immobilized cells and tissues with ∼20-50nm resolution and a field of view of ∼10-30µm resulting in near-native state imaging and the possibility of microscale, mesoscale and nanoscale correlative imaging. We have written a detailed protocol for optimization of FIB and SEM parameters to reduce imaging artefacts and enable downstream computational processing and analysis. While our experience is based on use of a single system, the protocol has been written to be as hardware and software agnostic as possible, with a focus on the purpose of each step rather than a fully procedural description to provide a useful resource regardless of the system/software in use.