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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 (150 kDa) to large viruses (2 MDa). For proteins smaller than 150 kDa the use of the Volta phase plate may be required.
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.
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 ~20° from the plane of the grid and are generally less than 300 nm thick.
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.
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