33.17:
Electron Microscope Tomography and Single-particle Reconstruction
The 3D structure of biological samples can be determined using two cryo-electron microscopy techniques — electron tomography and single-particle reconstruction.
In electron tomography, a sample is progressively tilted relative to the electron beam, capturing a series of 2D images from different angles. These 2D images are then stacked using computer software to generate the 3D image or tomogram of the sample.
Electron tomography maintains the in vivo structural integrity of the sample and is primarily used for 3D visualization of cells, organelles, and macromolecules in their native environment.
In single-particle reconstruction, isolated in vitro samples, such as a purified protein sample, are evenly distributed on a grid.
A 2D image of the individual protein molecules lying at random orientations is captured. Thousands of such images are then averaged and aligned to reconstruct the 3D image of the sample.
33.17:
Electron Microscope Tomography and Single-particle Reconstruction
Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
Electron Tomography
Electron tomography can be performed either in TEM or STEM (scanning transmission electron microscopy) mode. STEM is a technique primarily used for obtaining tomograms of thick biological specimens. It combines the sample surface scanning methodology of scanning electron microscopy, such that the electrons transmitted or scattered at each point where the electron beam hits the sample are collected by a series of detectors.
A variation of the conventional electron tomography technique is dual-axis tomography, where the sample is tilted around two axes with respect to the electron beam. This results in two separate tomograms that are then aligned to generate a 3D image of the sample. Dual-axis tomography has two advantages over single-axis tomography: a better reconstruction of the sample's expanded features and increased sample depth resolution.
Another variation is the electron cryo-tomography, in which samples are imaged under cryogenic conditions because fixation and dehydration can damage the biological structures. The method is primarily used for thin samples, less than 500 nm in thickness, because thick samples block the electron beam. Therefore, the technique has been limited to purified macromolecular complexes, viruses, or small cells such as bacterial cells.
Single-particle Reconstruction
Single-particle analysis is typically used with cryo-electron microscopy to generate 3D structures with near-atomic resolution. It is most suited for large or dynamic macromolecular complexes that are difficult to crystallize, making it a substitute for X-ray crystallography. For complexes that can be crystallized, single-particle analysis is a method of choice to obtain high-resolution structures. The technique has been used for studying membrane proteins, protein complexes, chromatin structure, and macromolecular machines such as ribosomes, and proteasomes.