Advanced cryo‐tomography workflow developments – correlative microscopy, milling automation and cryo‐lift‐out

Abstract

Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryofluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent

Cryo‐FIB preparation of whole cells and tissue for cryo‐TEM: use of high‐pressure frozen specimens in tubes and planchets

“…Many steps are involved, and each step is a potential source of difficulty. However, ongoing developments show that the field of cryo-TEM preparation is maturing into a more commonly used high-end technique (Kuba et al, 2020). Cryo-FIB-milled TEM lamellae provide unprecedented insights into native cellular environments.…”

“…Tremendous progress has been achieved since the technique was first published in 2006 (Marko et al, 2006), and the technique has evolved into a reliable route for detailed studies of macromolecular complexes within their native cellular environment (Marko et al, 2006;Wagner et al, 2017). TEM lift-out was long deemed 'practically impossible', but has emerged in two flavours: the classical lift-out with a cold needle (Parmenter et al, 2016;Kuba et al, 2020;Parmenter & Nizamudeen, 2020), and using a cold 'gripper' (Schaffer et al, 2019). TEM lift-out techniques also have the advantage of being part of existing workflows, in particular when loading into cryo-TEM'.…”

“…This sort of TEM, while very common, does not include the most popular type of high-end cryo-TEM, having a cartridgetype specimen holder. In that case, the cartridge itself is modified for use in both the FIB-SEM and the TEM (Kuba et al, 2020).…”

“…This can be complemented by SEM imaging of the surface of the sample as the FIB-milling proceeds. Changes in localised charge on the sample face can make cellular components visible (Schertel et al, 2013;Kuba et al, 2020), and this kind of image may be of use in computational comparison with a 3D light microscopic volume.…”

“…Since 2006, three approaches have emerged. (1) Cells or bacteria frozen on a TEM grid -the cryo-FIB thins areas of interest for cryo-TEM (Marko et al, 2006;Marko et al, 2007;Rigort et al, 2010;Strunk et al, 2012;Zhang et al, 2016;Schaffer et al, 2017); (2) the 'lift-out' technique, known from the semiconductor industry and mate-rial sciences (Mayer et al, 2007), but carried out under cryoconditions (Rubino et al, 2012;Mahamid et al, 2015;Parmenter et al, 2016;Schaffer et al, 2019;Kuba et al, 2020;Parmenter & Nizamudeen, 2020) and (3) freezing the specimen in a tube or planchet (also known as 'freezing hat' or 'platelet') -the cryo-FIB thins a region of the specimen using the 'H-bar' technique (Edwards et al, 2009;Hayles et al, 2010;Hsieh et al, 2014). The advantage of using planchets or tubes is the potential to investigate cell-cell contacts or specific organelles within cells in the context of surrounding cells, and in particular within tissue.…”