Single Particle Analysis (SPA)
Background
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.
Hardware and software
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.
SPA Cryo-Electron Microscopy Technique Overview
Source: National Cancer Institute U.S.A.
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. At NeCEN, automated dual-axis tomography is available at the two Titan Krios microscopes, enabling recording of two orthogonal tilt series, allowing many higher quality reconstructions. From the tilt series, three-dimensional reconstructions of the objects are then computed allowing unprecedented insight into biological structures.
Negative stain
Under construction