Main techniques: SPA and Cryo-ET
In general, the contrast in typical cryo-EM images is still very low. Therefore, depending on the sample and the specific biological question, several post-data collection image processing methods are necessary to improve the signal-to-noise ratio and to generate a 3D reconstruction from the 2D images. The two main techniques that are employed at NeCEN are single particle analysis (SPA) and cryo-electron tomography (cryo-ET). By combining many (sometimes thousands of) noisy images, high resolution structural details can be obtained.
SPA and cryo-ET allow scientists to zoom in on cells to the level of molecules and even atoms. The methods are suitable for a wide variety of research applications that can potentially lead to faster and better methods to understand, diagnose, cure, and prevent diseases at a molecular level.
Single Particle Analysis (SPA)
Structures of large proteins and macromolecular complexes can be determined to near-atomic resolution using a technique referred to as single particle analysis (SPA). From thousands of images containing two-dimensional projections of many individual three-dimensional molecular structures, information is deduced to reconstruct the underlying three-dimensional average structure. SPA also allows determination of various structural conformations if these are present in the sample.
SPA requires not only excellent high-resolution images, as produced by the cryo electron microscopes at NeCEN, but also knowledge and expertise to make use of software packages to extract the information from the images. Therefore the NeCEN microscopes are optimized for SPA with the most sensitive image detectors available together with automated data collection software.
Cryo-electron tomography (CET)
Cryo-electron tomography (CET) makes it possible to obtain a three-dimensional reconstruction of unique, heterogeneous biological structures, such as flexible macro-molecules, organelles, and small cells, when these are sufficiently thin to be imaged by transmission electron microscopy. In order to obtain a structure, a tilt series is recorded of a single specimen: a set of two-dimensional images recorded at different angles. From the tilt series, three-dimensional reconstructions of the objects are then computed allowing unprecedented insight into biological structures.