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

Abstract

SummaryThe desire to study macromolecular complexes within their cellular context requires the ability to produce thin samples suitable for cryo‐TEM (cryo‐transmission electron microscope) investigations. In this paper, we discuss two similar approaches, which were developed independently in Utrecht (the Netherlands) and Albany (USA). The methods are particularly suitable for both tissue samples and cell suspensions prepared by a high‐pressure freezer (HPF). The workflows are explained with particular attention to potential pitfalls, while underlying principles are highlighted (‘why to do so’). Although both workflows function with a high success rate, full execution requires considerable experience and remains demanding. In addition, throughput is low. We hope to encourage other research groups worldwide to take on the challenge of improving the HPF– cryo‐FIB‐SEM – cryo‐TEM workflow. We discuss a number of suggestions to this end.Lay DescriptionLife is ultimately dictated by the interaction of molecules in our bodies. Highly complex equipment is being used and further developed to study these interactions. The present paper describes methods to prepare small, very thin lamellae (area of 5×5 µm2, thickness 50–300 nm) of a cell to be studied in a cryo‐transmission electron microscope (cryo‐TEM). Special care must be taken to preserve the natural state of molecules in their natural environment. In the case of cryo‐TEM, the samples must be frozen and kept frozen to be compatible with the vacuum conditions in the microscope. The frozen condition imposes technical challenges which are addressed. Two approaches to obtain the thin lamellae are described. Both make use of a focused ion beam (FIB) microscope. The FIB allows removal of material with nanometre precision by focusing a beam of ionised atoms (gallium ions) onto the sample. Careful control of the FIB allows cutting out of the required thin lamellae. In both strategies, the thin lamellae remain attached to the original sample, and the ensemble of sample with section and sample holder is transported from the FIB microscope to the TEM while being kept frozen.