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Summary   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.    Lay Description  Life Sciences studies life down to the smallest details. Visualising the smallest details requires electron microscopy, which utilises high‐vacuum chambers. One method to maintain the integrity of Life Sciences samples under vacuum conditions is freezing. Frozen samples can remain in a suspended state. As a result, research can be carried out without having to change the chemistry or internal physical structure of the samples.  Two types of electron microscopes equipped with cryo‐sample handling facilities are used to investigate samples: The scanning electron microscope (SEM) which investigates surfaces and the transmission electron microscope (TEM) which investigates thin electron transparent sections (called lamellae). A third method of investigation combines a SEM with a focused ion beam (FIB) to form a cryo‐FIB‐SEM, which is the basis of this paper. The electron beam images the cryo‐sample surface while the ion beam mills into the surface to expose the interior of the sample. The latter is called cross‐sectioning and the result provides a way of investigating the 3rd dimension of the sample. This paper looks at the making of cross‐sections in this manner originating from knowledge and experience gained with this technique over many years. This information is meant for newcomers, and experienced researchers in cryo‐microscopy alike.

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