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This technique requires the collection of a large number of movies from mono-disperse protein complexes or viruses such that their 3D structure can be determined. The Titan Krios microscopes are equipped with direct electron detectors and automated data collection software that allow a large number of movies to be collected from single particle samples. The type of single particles that can be imaged range from small protein complexes (100 kDa) to large viruses (2 MDa). For proteins smaller than 100 kDa the use of the Volta phase plate may help with structure determination.
Molecular
This technique can be used with single particle samples and is excellent at generating initial models for single particle analysis or for analysing repeating structures in larger pleomorphic objects. Tilt series are collected at areas of interest, which can then be aligned and reconstructed to generate 3D volumes. For thicker specimens zero-loss imaging is recommended.
Cellular
This technique is used to look at large pleomorphic objects such as vesicles, isolated organelles, bacteria, and intact eukaryotic cells. Tilt series are collected at areas of interest which can then be aligned and reconstructed to generate 3D volumes. For thicker specimens zero-loss imaging is recommended and a maximum sample thickness is of 0.5 um is advised.
Microcrystal Electron Diffraction, or MicroED, is a valuable tool that delivers high-resolution data for a wide range of samples such as proteins, peptides, as well as organic molecules. It uses TEM under cryogenic temperature to collect electron diffraction data with high-speed and sensitive detectors under very low doses.
Electron crystallography requires thin 3D crystals or 2D crystals of lipid-embedded membrane proteins:
The primary use of the Scios and Aquilos cryoFIB/SEM is to generate lamellae for cryogenic TEM experiments (mainly tomography but also MicroED). Lamellae are thin sections of the target e.g. cells or crystals, that have been produced by milling away excess material in stages where the ion-beam current is reduced each time. This is done to avoid local heating whilst also increasing the milling rate. Typically, the lamellae are ~12° from the plane of the grid and are generally less than 300 nm thick.
The Helios plasmaFIB-SEM, jointly owned with the Rosalind Franklin Institute, is capable of using different ion sources from the traditional gallium of standard FIB-SEMs resulting in altered milling properties; these include greater milling speed and more delicate milling. One use of this is to perform serial-sectioning of an un-stained frozen-hydrated sample, including thick, high-pressure frozen tissue samples. The material is milled to expose a cross-section of the sample, then imaged with the SEM. The surface of this cross-section is then gently milled again to expose another surface deeper into the volume. The process is repeated until a 3D volume is built up, with x/y resolution on the order of 10s of nm, and z resolution ~50 nm, offering a view into the mesoscale of samples under a native-like hydration state.
This method employs the Leica cryoCLEM microscope to acquire mosaics of fluorescently labelled cryo-EM samples. The mosaics can then be utilised in ThermoFisher Scientific MAPS software or in SerialEM to guide cryo-ET data acquisition or cryoFIB lamella production. This method can help to identify regions of interest in frozen cryo-EM grids with the help of the fluorescence signals, before the cryo-EM session, vastly improving the efficiency of data collection/lamella preparation.
The chameleon is an instrument for preparing samples for single particle cryo-electron microscopy. The chameleon sprays the protein solution onto self-wicking TEM grids and plunges the grids into the cryogen. The sample dispense to vitrification time is significantly shorter than traditional blotting methods, such as the Vitrobot, down to 54ms. Grid preparation using the chameleon can improve sample quality for proteins that display preferred orientation, aggregation, or decomplexation at the air water interface.
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