High-throughput cryo-electron tomography enables multiscale visualization of the inner life of microbes.

Cryo-electron tomography (cryo-ET) is an advanced and rapidly evolving imaging technique that enables three-dimensional visualization of biological structures in their native state. Although cryo-ET has historically faced significant challenges, including limited applications, tedious data acquisition, labor-intensive image processing, and lower resolution when compared with single particle cryo-electron microscopy (cryo-EM), recent breakthroughs in hardware and software development have significantly improved the entire cryo-ET workflow to enable higher throughput and resolution. These advances have accelerated discoveries in structural and cellular biology, particularly in microbiology, where cryo-ET has unveiled unprecedented insights into the inner life of microbes. This review presents pivotal advances propelling high-throughput cryo-ET and the visualization of microbial architecture. As innovations in imaging technologies, workflow automation, and computational methods continue progressing rapidly, cryo-ET is expected to be increasingly utilized across various fields of life sciences, shaping the future of biological research and biomedical discoveries.