cryoEM
Cryo-Electron Microscopy (Cryo-EM) applies Transmission electron microscopy (TEM) to image particles, including macromolecular complexes, viruses, and cells in their native state at cryogenic temperatures as well as organic/inorganic materials.
Recent technological advances in sample preparation/vitrification strategies, computation and especially instrumentation (new image processing algorithms, Direct Electron Detection camera, Volta phase plate) allowed Cryo-EM single particle analysis (Cryo-EM SPA) to become a newly dominant discipline, accompanied by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. It has prompted revolution in structural biology, allowing researchers to use Cryo-EM to solve near-atomic-resolution macromolecular structures.
In Cryo-EM SPA, three-dimensional density maps can be reconstructed by averaging a large number of two-dimensional projection images of the same object along different directions. It covers a wide molecular weight range of specimens, from protein complexes in the tens of kDa to large virus particles with hundreds of mDa. Moreover regular fibrillar protein aggregates (amyloids) have also been successfully targeted through cryo EM, further highlighting the impact of this approach on biological and biomedical research.
The inter-departmental Florence Center for Electron Nanoscopy (FloCEN) houses state-of-the-art equipment for cryo-EM. This includes a ThermoFisher Glacios at 200-kV, also equipped with a Falcon III direction electron detector, and a ThermoFisher Vitrobot Mark IV for specimen preparation.
EM specimens are typically prepared using 3 μl sample solution at a concentration of 0.05 – 5 μM
To be completed...
References
- Rebecca F. Thompson, Matt Walker, C. Alistair Siebert, Stephen P. Muench, Neil A. Ranson, An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology, Methods, Volume 100, 1 May 2016, Pages 3-15, https://doi.org/10.1016/j.ymeth.2016.02.017.
- Lori A. Passmore, and Christopher J. Russo, Specimen preparation for high-resolution cryo-EM, Methods Enzymol. 2016; 579: 51–86, https://dx.doi.org/10.1016%2Fbs.mie.2016.04.011