Material sizes accessible to 3DEM methods range from tens of nanometers to hundreds of millimeters. Example specimens from left to right: virus, algae cell, worm, fish embryo, human brain slice. (Fixation) Different methods are selected based on the size of the specimen. Vitrification preserves the most native ultrastructure. However, plunge freezing is limited to cells smaller than 10 m, and high-pressure freezing is only effective for samples thinner than 200 m. Larger samples may require chemical fixation (preceded by vibratome sectioning for thick tissue), dehydration, and resin embedding, which cause some ultrastructural distortion and loss of high-resolution detail. (Thinning) This step is necessary for most cellular samples. It is performed on vitrified or resin-embedded samples using focused ion beams (FIBs) or sharp diamond knives. While FIB milling yields compression-free slices with the highest structural preservation, diamond knives allow serial sectioning. Not pictured: CEMOVIS diamond knife sectioning of HPF vitreous samples, which is less routinely practiced in current workflows. (Acquisition) The final 3DEM imaging mode, along with the type of upstream processing (e.g., ice or resin), determines the obtainable resolution and maximum continuous volume. Transmission electron microscopy (TEM) currently provides the highest resolution, scanning TEM (STEM) tomography covers larger volumes at the trade-off of resolution, and scanning electron microscopy (SEM) of block faces sequentially thinned by FIB or diamond knife provides access to the largest continuous volumes. In blue and orange brackets, we indicate the acquisition modes discussed in this Review for cryo-ET and vEM. Although here we define cryo-ET as TEM imaging (as is most common in the field), cryo-STEM tomography can be considered a distinct mode of cryo-ET. Room temperature TEM and STEM tomography have a long history of use for resin-embedded cells and tissue but are not covered in this Review.
