3D reconstruction of biological structures: automated procedures for alignment and reconstruction of multiple tilt series in electron tomography.

Authors:
Jason Lanman
Jason Lanman
Purdue
Assistant Professor
EM tomography
West Lafayette, IN | United States

Adv Struct Chem Imaging 2017 28;2. Epub 2016 Jun 28.

National Center For Microscopy and Imaging Research, Center for Research in Biological Systems, University of California, 9500 Gilman Drive La Jolla, San Diego, CA 92093-0608, USA.

Transmission electron microscopy allows the collection of multiple views of specimens and their computerized three-dimensional reconstruction and analysis with electron tomography. Here we describe development of methods for automated multi-tilt data acquisition, tilt-series processing, and alignment which allow assembly of electron tomographic data from a greater number of tilt series, yielding enhanced data quality and increasing contrast associated with weakly stained structures. This scheme facilitates visualization of nanometer scale details of fine structure in volumes taken from plastic-embedded samples of biological specimens in all dimensions. As heavy metal-contrasted plastic-embedded samples are less sensitive to the overall dose rather than the electron dose rate, an optimal resampling of the reconstruction space can be achieved by accumulating lower dose electron micrographs of the same area over a wider range of specimen orientations. The computerized multiple tilt series collection scheme is implemented together with automated advanced procedures making collection, image alignment, and processing of multi-tilt tomography data a seamless process. We demonstrate high-quality reconstructions from samples of well-described biological structures. These include the giant Mimivirus and clathrin-coated vesicles, imaged in situ in their normal intracellular contexts. Examples are provided from samples of cultured cells prepared by high-pressure freezing and freeze-substitution as well as by chemical fixation before epoxy resin embedding.

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http://dx.doi.org/10.1186/s40679-016-0021-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972035PMC
June 2016
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4 Citations

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