Cryo-focused ion beam (cryo-FIB) milling of frozen hydrated cells to produce thin lamellas in combination with cryo-electron tomography (cryo-ET) has led to unprecedented insights into the cell interior. This method opens the door to native structures deep inside the cells and makes structural investigations of macromolecules in situ possible. Various implementations of cryo-FIB milling for cells deposited or grown directly on EM grids have been realized, and some preliminary results suggest that this approach can be extended to high-pressure frozen tissue. However, a small volume of interest must be extracted from a bulky sample, i.e. a process analogous to a clinical biopsy, which can be performed under cryogenic conditions.
While the preparation process must be specially tailored to the peculiar properties of the sample, it is also necessary to adapt the tomographic recording and analysis process to both the sample and the respective research question. Unfortunately, there is no universal recipe, neither for sample preparation nor for data acquisition, and neither for unlocking and decoding the information content of tomograms, that is suitable for each individual sample.
In this lecture, we will present our recent work in the field of cryo-ET and in situ structural biology, highlighting technological developments and their potential, and offering a perspective to obtain "anatomical" details at the molecular level from larger cells or tissues - towards a biopsy at the nanoscale.