Electronic cryo-tomography ( ECT , also cryo-electron tomography, cryo-ET or CET ) is a method of obtaining high-resolution three-dimensional images (~ 4 nm). Used to obtain images of biological macromolecules and cells [1] .
ECT is used using transmission electron microscopy (TEM), in which the samples are tilted at different angles to the electron beam, resulting in a series of two-dimensional images. A series of tilted two-dimensional images are processed on a computer, resulting in a three-dimensional tomogram.
Unlike methods using electronic tomography , in this method the test samples are frozen using a special technology so that the test object is not damaged by ice crystals, pressure, chemicals and other factors. This procedure is called cryofixation. Usually, the organic sample is cooled so that the ice formed is amorphous (non-crystalline, that is, vitrification is carried out) [2] , and transmission is performed under cryogenic conditions at temperatures below ° C, which prevents the destruction of biological structures [3] .
Description of technology
In electron microscopy (EM), the samples are in a deep vacuum. Such a vacuum is not applicable for biological samples, since water boils in the cells and they explode. At room temperature in EM, the samples are dehydrated. Another approach to stabilizing biological samples is to freeze them ( cryo-electron microscopy ). In cryo-electron microscopy, samples (usually small cells (e.g., bacteria or Archaea ) or viruses ) are prepared for examination in ordinary aqueous media. Samples are immersed in a cryogen (usually liquid ethane ), while the water molecules do not have time to rebuild into a crystal lattice. As a result of such cooling, the water becomes amorphous ice. [2] At the same time, cellular structures such as lipid membranes, which usually break down during normal freezing, are preserved. Frozen samples are stored at liquid nitrogen temperature and the water does not warm up enough to crystallize.
Samples are viewed in a transmission electron microscope (TEM). They tilt at different angles with respect to the electron beam (usually every 1 or 2 degrees from -60 ° to + 60 °), images are obtained at each angle. A series of images is processed on a computer and a three-dimensional image of the object of interest is obtained [4] . The resulting image is called a tomogram or tomographic reconstruction.
Features
In transmission electron microscopy (TEM), electrons interact with the material of a sample, so resolution is limited by its thickness. Samples must have a thickness of at least ~ 500 nm to achieve a “macromolecular” resolution (~ 4 nm). For this reason, most ECT studies have focused on the study of purified macromolecular complexes, viruses, and small cells, such as many types of bacteria and Archaea .
Strong interaction of electrons with matter leads to anisotropy effects. When the sample is tilted, the electron beam interacts with a relatively large cross-sectional area. This leads to the fact that in practice, tilt angles greater than 60-70 ° do not provide much information and therefore are not used.
Cryofluorescence microscopy [5] , light microscopy (for example, cryo-Palm [6] ) and other techniques are also used in ECT. In these techniques, a sample containing a fluorescently labeled protein is frozen and viewed under a light microscope. In this case, the sample should be stored at temperatures (below -150 ° C). A fluorescent signal is identified and the sample is transferred for study in cryo-ET.
See also
- Cryo-electron microscopy
- Electron microscopy
- Electron tomography
- Transmission electron microscopy
Notes
- ↑ Gan, Lu; Jensen, Grant J. Electron tomography of cells (neopr.) // Quarterly Reviews of Biophysics. - 2012. - 1 February ( t. 45 , No. 1 ). - S. 27-56 . - ISSN 1469-8994 . - DOI : 10.1017 / S0033583511000102 . - PMID 22082691 .
- ↑ 1 2 http://www.nsu.ru/xmlui/bitstream/handle/nsu/589/EM-in-cytology.pdf
- ↑ Dubochet, J .; Adrian, M .; Chang, JJ; Homo, JC; Lepault, J .; McDowall, AW; Schultz, P. Cryo-electron microscopy of vitrified specimens (neopr.) // Quarterly Reviews of Biophysics. - 1988. - 1 May ( t. 21 , No. 2 ). - S. 129-228 . - ISSN 0033-5835 . - DOI : 10.1017 / s0033583500004297 . - PMID 3043536 .
- ↑ Lučič, Vladan; Rigort, Alexander; Baumeister, Wolfgang. Cryo-electron tomography: the challenge of doing structural biology in situ (English) // The Journal of Cell Biology : journal. - 2013 .-- 5 August ( vol. 202 , no. 3 ). - P. 407-419 . - ISSN 1540-8140 . - DOI : 10.1083 / jcb.201304193 . - PMID 23918936 .
- ↑ Zhang, Peijun. Correlative cryo-electron tomography and optical microscopy of cells (English) // Current Opinion in Structural Biology: journal. - 2013 .-- 1 October ( vol. 23 , no. 5 ). - P. 763-770 . - ISSN 1879-033X . - DOI : 10.1016 / j.sbi.2013.07.07.017 . - PMID 23962486 .
- ↑ Chang, Yi-Wei. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography (Eng.) // Nature Methods : journal. - 2014 .-- 1 July ( vol. 11 , no. 7 ). - P. 737-739 . - ISSN 1548-7105 . - DOI : 10.1038 / nmeth . 2961 . - PMID 24813625 .