Automated cryo-volume EM for high-resolution 3D imaging and in situ structural analysis of cells and tissues

Cryo-volume electron microscopy (CVEM) enables three-dimensional imaging of biological ultrastructure in a near-native state but has been limited by low image contrast and charging artifacts that hinder data interpretation and complicate automation of data acquisition. Here we present an experimental and computational workflow that combines orthogonal cryo-SEM imaging, spot-geometry optimized O+ plasma-FIB milling, dedicated acquisition-control routines, and dedicated image alignment procedure. The workflow enables autonomous acquisition of volumetric datasets from vitrified cells and tissues at ∼15–20 nm isotropic resolution. In addition, sub-volume averaging of 113 nuclear pore complexes extracted from CVEM dataset of Cos-7 cell yielded its reconstruction at 9.4 nm resolution. Together, these results establish CVEM as a robust platform for autonomous high-resolution volumetric imaging and structural analysis of vitrified biological specimens.