Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography

Abstract

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples.

Figure 1. AutoGrids modified for cryo-FIB.

a) Top and b) bottom side of an AutoGrid holding a clipped TEM grid. The flat top side shows the cutout required for cryo-FIB milling. c) The FIB shuttle loaded with two AutoGrids. Detailed view showing a properly oriented AutoGrid loaded into the shuttle. Note that the AutoGrid cutout faces up.

Figure 2. Sample clipping and loading.

a-c) The sample loading box with double-walled reservoir (9). The second reservoir is filled with liquid nitrogen through the filling gaps (2, 8). The AutoGrid notch (6) aids the fast and easy handling of Autogrids under liquid nitrogen. The clipping tool (1) and tweezers can be cooled in the round bore (7). The shuttle (3) (in loading position), sample storage box (4) and clipping metal support (5) are immersed under liquid nitrogen.

Figure 3. Sample transfer system.

a) The Quorum cryo-system with the transfer unit (1). The pumping station (2) with the loading box inside. b) The sample transfer unit with the small transfer chamber (3) and the shuttle (4) attached to the transfer rod. c) The loading box (5) inside the pumping station (7). Here, the shuttle is in the transfer position (6). d) The transfer unit attached to the loading box for shuttle pick-up/drop-off. The small transfer chamber (3) is closed/opened using the transfer unit valve (8).

Figure 4. Lamella milling.

a). Scanning electron microscope (SEM) image of an AutoGrid aligned with the incident direction of the ion beam. b). The AutoGrid is centered at the GIS position in the SEM image and the GIS needle is inserted. c). SEM image of a TEM grid with Chlamydomonas cells. d). SEM image of two target Chlamydomonas cells before FIB milling. e). FIB-induced secondary electron (FIB SE) image of the two target cells with two rectangle standard milling patterns as a starting point for the milling procedure. f). FIB SE image showing the milling step at 50pA where patterns on both sides are made slimmer (but pattern width remains constant) and shifted closer to the lamella edge. g, i). FIB SE and h) SEM images of the finished lamella after cryo-FIB milling. See Video 1 for an overview of cryo-FIB milling.

Figure 5. TEM images of final lamellas.

a) Cross-section of a Chlamydomonas cell, with a lamella thickness of 100 nm. b) Cross-section through several yeast cells, with a lamella thickness of 250 nm.