Cryo-Tomography of Vitrified Bacterial and Human Cells by Scanning Transmission Electron Microscopy

Abstract

Cryo-transmission electron tomography (CET) has emerged as a vital tool for structural biology studies of cells and viruses. Direct imaging of fully hydrated, vitrified material represents the state of the art for preservation of biological samples. Lacking heavy metal stains, CET relies on phase contrast typically obtained by defocusing the sample. The dependence on phase coherence, as well as cumulative radiation damage on frozen hydrated specimens, impose an inherent upper limit on sample thickness and usable tilt range. Even with energy filtration to remove the contribution of inelastic scattering, CET suffers from missing wedge effects and low signal-to-noise ratio. Scanning transmission electron microscopy (STEM) circumvents the need for phase contrast with incoherent detection, and has recently been extended to biological tomography. However the weak electron scattering by light elements was thought to preclude its application to unstained cryogenic specimens. We show this not to be the case. To the contrary, we find that raster scanning permits a higher total dose than conventional wide-field imaging, and the independent STEM detection in bright and dark field detectors provides sufficient contrast to show detailed cellular architecture similar to that provided by wide-field tomography. An important difference is that the specimen remains dynamically in focus even at high very tilts up to 70°. This significantly improves the depth resolution in reconstructions. Sample thickness limitations are also relaxed. We demonstrate the cryo-STEM tomography (CSTET) method using unstained, vitrified bacteria and human epithelial cells.