Cryo-Electron Tomography Technology

Cellular cryo-electron tomography (cryo-ET) is a label-free cryogenic imaging technique that provides 3D datasets of organelles and protein complexes at nanometer resolution in their physiological environments. This is done by opening windows into the cell with focused ion beam (FIB) milling of a cryogenically frozen (vitrified) cell. A series of 2D images is taken of this thinned cellular sample (cryo-lamella) then reconstructed into a 3D dataset. Such high-resolution 3D images of the interior of cells provide new insights into cellular function and sheds light on the arrangement and structure of native protein complexes.

Cryo-electron tomography has been applied to many different sample types, from single molecules, to protein complexes, viruses, bacteria, and cells, to tissue cells and large tissue samples. Cells are either vitrified through plunge-freezing (as in single particle analysis) or high-pressure freezing (HPF). The water in the sample freezes rapidly and does not crystallize, thus avoiding the molecular-scale disruption (caused by ice crystal formation) that would occur with a normal slow freezing process.  Samples are carried on grids, which are designed for the transmission electron microscope. Depending on the size of the sample, plunge freezing is all that is needed.  However, for larger samples like cells or tissues, samples must be thinned by cryo-FIB milling so that electrons can pass through the sample.

Cryo-ET is used to obtain 3D information from vitrified samples. Depending on the size of the sample, a thinning step is included in the sample preparation using focused ion beam (FIB) or plasma ion beam milling to produce cryo-lamella that can be penetrated by electron beams. 

Light microscopy, particularly fluorescence microscopy, is a cornerstone of modern cell biology. Advances in optics, as well as computational tools, have made it possible to view systems in a range of relevant scales. It allows for the identification of proteins and molecules based on bound fluorescent tags. Structures that are not fluorescently labeled, however, cannot be visualized, meaning the context surrounding the fluorescent label is lost. Moreover, the presence of certain chemical agents needed for some fluorescence modalities to work can also affect the native structure. Locating the structure of interest can be difficult in the vast complexity of the natural cellular environment. However, using cryo-correlative microscopy (cryo-CLEM), the structures of interest are identified in the cryo-fluorescence light microscope. The iFLM (Integrated Fluorescence Light Microscope) Correlative System delivers the localization of fluorescent targets inside the chamber, allowing users to check the fluorescence signal inside milled lamellae, eliminating extra sample transfer steps and correlate two imaging modalities directly within one system. The iFLM Correlative System is available on the Thermo Scientific Aquilos 2 Cryo-FIB, Thermo Scientific Helios Hydra Cryo-Plasma-FIB, and Thermo Scientific Arctis Cryo-Plasma-FIB.

Reliable results from cryo-ET rely on high-quality cryo-lamella preparation. When used with light microscopy, which provides the location of a labeled protein of interest, cryo-ET brings together the best of both worlds: localization, specificity, and nanometer resolution. A new integrated cryo-ET workflow from Thermo Fisher Scientific illustrates how these two techniques can be combined to achieve results.