Tim Laugks

Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography

Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

Micromachining tools and correlative approaches for cellular cryo-electron tomography.

A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor. An alternative approach that avoids mechanical deformations is the use of focused ion beam (FIB) instrumentation, where thinning of the frozen-hydrated specimen occurs through the process of sputtering with heavy ions, typically gallium. Here, we use correlative cryo-fluorescence microscopy to navigate large cellular volumes and to localize specific cellular targets. We show that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling. We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography.

Optimized cryo-focused ion beam sample preparation aimed at in situ structural studies of membrane proteins

While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution.

In Situ Tomography of Membrane Proteins Enabled by Advanced Cryo-FIB Sample Preparation and Phase Plate Imaging

The development of cryo-focused ion beam (cryo-FIB) microscopy into a highly reliable sample preparation technique for frozen-hydrated specimens has recently enabled cryo-electron tomography (CET) of eukaryotic cells with unprecedented resolution and image quality [1,2,3]. The ability to prepare distortion-free lamellas of homogenous, user-defined thickness has allowed in situ studies of cellular structures in their native state, revealing new insights into biological processes [4]. Additionally, cryo-fluorescence microscopy has been combined with cryo-FIB for the in situ targeting of fluorescently-labelled cellular structures [5].

Cryo-FIB Lift-out Sample Preparation Using a Novel Cryo-gripper Tool

In recent years, biological specimen preparation using a cryo-focused ion beam (cryo-FIB) has become a key technique for investigating complex cellular structures in situ [1-3]. Cryo-FIB has enabled cryoelectron tomography (cryo-ET) of plunge-frozen hydrated cells, revealing the cellular interior with sufficient resolution and contrast to study membrane-bound macromolecules in their native state [4-7].