In cryo-ET, tilt-series acquisition of an ROI involves incrementally tilting the cryo-preserved specimen along one or two axes 13 while a series of projection images are captured on a detector. However, difficulties remain for bridging the disparity of spatial scales in multimodal microscopy pipelines where the field of view (FOV) in wide-field FLM can be 10 5 times (~0.1 to 5 mm) that of an EM image (~200 nm to 800 μm) 11, 12. In combination with cryo-FIB milling, it is now possible to pinpoint an area of interest deep in the interior of a specimen with 3D correlative cryo-FLM-FIB-ET 9, 10. Cryo-correlative light and electron microscopy (cryo-CLEM) 8 directly connects temporal and spatial information from fluorescence light microscopy (FLM) with cryo-ET ultrastructural data of a region of interest (ROI). To explore thicker regions of cells, sample thinning technologies have evolved and include cryo-electron microscopy of vitreous sections 6 and cryo-focused-ion-beam (cryo-FIB) milling 7, each of which may introduce artifacts to the sample. Cryo-ET is generally restricted to investigations of small specimen volumes and the thin peripheral areas of cells (<500 nm) that are penetrable by the electron beam. Computationally extracted sub-tomograms can be averaged and classified to reveal sub-nanometer to nanometer resolution (3 Å to 4 nm) structures of in situ complexes 3, 4, 5. Cryo-electron tomography (cryo-ET) links three-dimensional (3D) contextual visualization and high-resolution structure determination of cryogenically preserved macromolecules in their native cellular environment 2. Cryo-electron microscopy (cryo-EM) of purified proteins (for example, single-particle cryo-EM) has propelled forward the cryo-EM ‘resolution revolution’ 1 resulting in an increasing interest in technologies to enable structure–function studies of macromolecules within the framework of larger biological systems.
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