Cryo‐FIB‐lift‐out: practically impossible to practical reality

Abstract

In this paper, we explore the development of the Cryo-Lift-Out (cryo-LO) technique as preparation tool for cryogenic transmission electron microscopy (cryo-TEM). What started in early work defying what was considered 'practically impossible' has developed into state-of-the-art practical reality. This paper presents the key hardware, basic principles and key considerations for the practical usage of cryogenic Lift-Out for those new to the field. Detailed protocols and in-depth description of considerations and points for further development are presented. The authors have attempted to formalise everything known about the technique gathered together from their expertise gained in the development of this approach.

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

“…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'.…”

“…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.…”

An introduction to cryo‐FIB‐SEM cross‐sectioning of frozen, hydrated Life Science samples

“…This success is the result of the combined efforts and determination of skilled mechanical and electrical engineers, physicists and biologists, facilitated by the development of high-end cryo-FIB-SEM and cryo-TEM instruments. Various practical approaches are proven to work, as discussed elsewhere in this special issue (Kuba et al, 2020;Parmenter, 2020;De Winter et al, 2020). Moreover, new biological insights have been obtained with the new techniques.…”

Recent Advances in Single-Particle Electron Microscopic Analysis of Autophagy Degradation Machinery

“…An approach that can overcome this challenge is cryo-focused ion beam milling or cryoFIB, which uses a gallium ion beam to generate thin lamellae specimens from vitrified cell samples as cryo-focused ion beam milling [129][130][131][132][133]. The recent improvement in instrumentation and experimental workflow have dramatically improved the success and throughput in producing high-quality thin specimens [134][135][136]. Specimens obtained from cryoFIB can then be transferred to a transmission electron microscope to be examined by cryo-electron tomography (cryo-ET), which involves acquiring a series of images of the specimens at different tilt angles.…”