Cryo-FIB-enabled 3D imaging of biological specimens.

Cryo-electron tomography (cryo-ET) has become a key technique for observing the three-dimensional structures of biomolecular complexes and organelles in cellular and tissue environments in situ. However, for thicker specimens that cannot be directly imaged at an electron-transparent thickness, the practical success of cryo-ET depends largely on workflow design that reproducibly yields high-quality, target-containing lamellae. Cryo-FIB-SEM is the de facto standard platform for precisely machining frozen specimens to an electron-transparent thickness, and it integrates the overall process into a single system, encompassing targeting via cryo-CLEM, the management of major failure modes such as contamination, charging, curtaining, and downstream steps for data reconstruction and interpretation. This review summarizes a cryo-FIB-SEM–centered cryo-ET workflow from the perspectives of specimen preparation, targeting, milling, acquisition, reconstruction, and interpretation. In addition, we discuss extended preparation and imaging strategies for thicker specimens, including high-pressure freezing (HPF), the waffle method, cryo-FIB-SEM slice-and-view imaging, and Serial Lift-Out, highlighting both their expanding capabilities and the operational challenges that remain for robust and scalable implementation.