Cryo Electron Microscopy (Cryo-EM)

For biological macromolecules such as proteins, cryo-EM now allows direct structural determination to near atomic resolution. This is achieved by imaging many thousands of individual protein molecules which can then be averaged to obtain high resolution information (single-particle reconstruction). 

Because cryo-EM images of proteins can capture heterogeneity within the molecular population, it is possible to carry out reconstructions of proteins in different conformational states, for example with and without a ligand bound.

Imaging structures larger than proteins, which may often be present in low or single copy numbers can also be carried out using cryo-EM. Various approaches can be taken, but an increasingly popular option is cryo-electron tomography (cryo-ET). In this, the sample is rotated in the microscope, and images taken at different rotation angles are used to reconstruct a 3-dimensional volume. The resolution of such reconstructions is usually lower than those obtained using the single-particle approaches, but in favourable cases, atomic structures can be fitted to cryo-ET reconstructions, or in some cases directly determined if the target is present as multiple copies.

Often, imaging larger cellular structures requires thinning the sample sufficiently to allow penetration by the electron beam. This can be carried out using a cryo-ultramicrotome, or by milling the sample using an ion beam in a scanning electron microscope (FIB-SEM). 

A particular challenge can be locating the region of interest within the sample. One way to address this is to fluorescently label the molecule of interest and visualise the sample at low magnification in a light microscope. The resulting images can then be used to map the target in higher magnification EM images, so called correlative light-electron microscopy (CLEM).

Our core activities are built around cryogenic single-particle imaging to determine macromolecular structures. We can provide assistance and training for all steps of the process including sample preparation, imaging, reconstruction, model building and analysis. 

We can also offer imaging of larger structures such as extracellular vesicles or liposomes suitably prepared.

A full list of SBC equipment is available. 

We are currently working to establish cryo-ET and CLEM approaches in the core in conjunction with the University FIB-SEM facility.

Please contact us if you think your project would benefit from such approaches.