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

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.