Dose-Efficient Cryo-Electron Microscopy for Thick Samples using Tilt-Corrected Scanning Transmission Electron Microscopy, Demonstrated on Cells and Single Particles

Cryo-EM is a powerful tool in structural biology, providing insights through techniques like single-particle analysis (SPA) and cryogenic electron tomography (cryo-ET). In thick specimens, challenges arise as an exponentially larger fraction of the transmitted electrons lose energy from inelastic scattering and can no longer be properly focused as a result of chromatic aberrations in the post-specimen optics. Rather than filtering out the inelastic scattering at the price of reducing potential signal, as is done in energy-filtered transmission electron microscopy (EFTEM), we show how a dose-efficient and unfiltered image can be rapidly obtained using tilt-corrected bright-field scanning-TEM (tcBF-STEM) data collected on a pixelated detector. Enhanced contrast and a 3-5x improvement in collection efficiency are observed for 2D images of intact bacterial cells and large organelles using tcBF-STEM compared to EFTEM for thicknesses beyond 500 nm. As a proof of concept for the technique’s performance in structural determination, we present an SPA map at a 7 Å nominal resolution for a highly symmetric virus-like particle (VLP) with 789 particles. These findings suggest applications for tcBF-STEM in cryo-EM of thicker cellular volumes where current approaches struggle.