A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.

Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing.

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.

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

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.

Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy.

The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process.

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.

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.