Cryo-electron tomography of large biological specimens vitrified by plunge freezing

Abstract

Cryo-focused ion beam (cryo-FIB) milling allows thinning vitrified cells for high resolution imaging by cryo-electron tomography (cryo-ET). However, it remains challenging to apply this workflow to voluminous biological specimens such as tissues or particularly large mammalian cells, which usually require high-pressure freezing for vitrification. Here we show that adult mouse cardiomyocytes and dissected Drosophila tissues can be directly vitrified by plunge freezing upon a short incubation in 10% glycerol. This expedites subsequent cryo-FIB/ET, enabling systematic analyses of the molecular architecture of complex native specimens. Our data provides unanticipated insights into the molecular architecture of samples hitherto unexplored by cryo-ET.