VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography

Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently pre -paring frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. The workflow includes vibratome slicing, high-pressure freezing, ultramicrotome cryo-trimming and cryo-FIB milling. Two strategies were developed for loading cryo-lamella via a side-entry cryo-holder or an FEI AutoGrid. The workflow was validated by using various tissue specimens, including rat skeletal muscle, rat liver and spinach leaf spec-imens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples.

Automated summary

This study reports an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella for cryo-electron tomography studies. The workflow was validated by obtaining in situ structures of ribosomes from spinach and liver samples.