Cryo-TEM – electron tomography and single particle analysis (SPA)

About this technique

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Electron Tomography

Electron tomography (ET) is ideally employed in conjunction with biological specimens that have been prepared using a fast-freezing/freeze-fixation approach, so that the physiological and functional state and structure of the sample is preserved as reliably as possible prior to image acquisition. In the case of electron tomography of cells for instance, immobilising all cellular processes near instantaneously (i.e. within just a few milliseconds) allows delicate structures and dynamic events to be captured in a ‘close-to-native’ state. Typically, the frozen-hydrated cells are then processed for transmission electron microscopy TEM) at ultra-low temperatures using a method called 'freeze-substitution', and subsequently infiltrated with one of several resins commonly used during conventional TEM specimen preparation.

Once embedded in plastic, the cells/tissue are sliced into relatively thick sequential sections ranging in thickness from 200 nm to 400 nm. These are then viewed either individually or in series. Thick sections rather than conventional thin (60-100 nm) sections are cut to visualise as much of the depth of the specimen as quickly as possible and to minimise disruption of the tissue by reducing the number of times the specimen has to be physically sectioned. 2-D images are then digitally collected using a TEM that operates at higher-than-normal voltages (e.g. ≥200 keV compared to conventional EMs that operate in the range of 80-120 keV), as the section is serially tilted by small, regular increments (e.g. 1° or 1.5°) over a relatively large angular tilt range (e.g. ±60-70°).

The image sets  – referred to as a 'tilt series' - are first aligned with one another using a mathematical technique called 'cross-correlation', which uses information that is common to each image to bring them crudely into register. These pre-aligned images are then more accurately aligned by tracking the positions of small gold fiducial markers placed on the top and bottom of each section prior to imaging in the TEM. A 3D density distribution or tomogram is computed from each set of aligned tilts around a single axis using another mathematical technique referred to as weighted back-projection. For cellular tomography, tilt series data are preferentially collected around two orthogonal axes rather than just a single axis because the resolution of an object within the plane of a specimen having a slab geometry such as a thick plastic section also depends on its orientation relative to the axis around which the specimen is tilted. Individual tomograms generated for each axis are brought into register and then combined in 3D space to yield a final dual-axis reconstruction that exhibits improved symmetry and resolution in all three dimensions. The resulting 3D cellular reconstructions can then be carefully 'segmented' for image analysis by outlining each object of interest to produce a precise 3D map for visualisation and annotation that accurately depicts subcellular organisation at high resolution (4-10 nm).

Single Particle Analysis

Another 3D imaging approach that falls under the ET banner is the method known as single particle analysis (SPA). SPA provides a critical resolution bridge between high-end structural techniques such as X-ray crystallography and nuclear magnetic resonance (NMR), and cellular tomography. SPA allows single molecules as well as macromolecular assemblies to be imaged then reconstructed at relatively high resolution (down to a few Ângstoms) for detailed analysis of their 3D structure. Although SPA can also be used in combination with the acquisition of a limited tilt series of 2D images taken as the specimen is tilted at different angles in the TEM, SPA fundamentally relies on using the large number of identical particles, oriented randomly and captured in each field of view to compute a 3D model of the particle structure.

The ideal scenario for structural studies by SPA is to first immobilse the purified preparation of molecules or protein complexes onto TEM specimen supports (grids) using a fast-freezing approach like 'plunge-freezing', which permits the ultra-rapid rates of cooling required to achieve sample vitrification without the formation of crystalline ice. The composite 3D image is produced by first sorting the arbitrarily oriented particles into different classes based on the different orientations of the particles on the grid. The examples obtained for each different class are then averaged (class-averaging) to generate a composite image for each class that now displays a relatively high signal-to-noise ratio. All of these composite images representing different views of the particle in various orientations are finally combined to compute an overall 3D model. SPA is particularly well suited to structures that display a high degree of symmetry, as is the case for a large number of viruses.

MicroED is an emerging technique that offers alternative opportunities to elucidate the structure of proteins and small molecules. It uses much smaller crystals than traditional X-ray diffraction.