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

Abstract

The introduction of cryo-techniques to the focused ion-beam scanning electron microscope (FIB-SEM) has brought new opportunities to study frozen, hydrated samples from the field of Life Sciences. Cryo-techniques have long been employed in electron microscopy. Thin electron transparent sections are produced by cryo-ultramicrotomy for observation in a cryo-transmission electron microscope (TEM). Cryo-TEM is presently reaching the imaging of macromolecular structures. In parallel, cryo-fractured surfaces from bulk materials have been investigated by cryo-SEM. Both cryo-TEM and cryo-SEM have provided a wealth of information, despite being 2D techniques. Cryo-TEM tomography does provide 3D information, but the thickness of the volume has a maximum of 200-300 nm, which limits the 3D information within the context of specific structures. FIB-milling enables imaging additional planes by creating cross-sections (e.g. cross-sectioning or site-specific X-sectioning) perpendicular to the cryo-fracture surface, thus adding a third imaging dimension to the cryo-SEM. This paper discusses how to produce suitable cryo-FIB-SEM cross-section results from frozen, hydrated Life Science samples with emphasis on 'common knowledge' and reoccurring observations.

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

“…Once the lamella has been extracted, attached to the support grid and thinned, the lamella is ready for transfer to a precooled cryo-TEM holder. Transfer to a storage facility is also an option, but poses the risk for the lamella to collect contamination from liquid nitrogen-born ice balls (Hayles et al, 2020).…”

“…Ultimately the sample must remain below -138°C and the anti-contaminator is kept 20-30°C lower. A much colder anti-contaminator will lower the partial water vapour pressure in the microscope chamber which increases the sublimation rate of the sample (Hayles et al, 2020). This is in particular potentially risky for a 100 nm thin lamella that remains in the chamber for many hours.…”

“…Vitrification of hydrated Life Science samples is crucial in order to preserve the structures down to the macromolecular complexes level (Hayles et al, 2020). Vitrification slows down water molecules into a solid phase.…”

“…A lower water vapour pressure reduces contamination on the surface of the cryo-TEM lamella. However, a too low temperature on the anti-contaminator, that is a too low water vapour pressure will sublime water from the lamella, potentially affecting the lamella (Hayles et al, 2020). This is also true once the lamella has been fixed onto the support grid.…”

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

“…Some substance is usually added, which functions as a 'filler' or 'cryo-protectant', and ideally both (McDonald, 2007;Möbius et al, 2010;Mielanczyk et al, 2014). A filler is used to avoid imperfect and inhomogeneous freezing from voids of air, while a cryo-protectant binds to water molecules, preventing ice crystallisation (Hayles & De Winter, 2020).…”