title: 3D reconstruction of biological structures: automated procedures for alignment and reconstruction of multiple tilt series in electron tomography.
abstract: 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.
authors.affiliation: 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.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Phan
authors.affiliation: 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.
authors.forename: Daniela
authors.initials: D
authors.lastname: Boassa
authors.affiliation: 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.
authors.forename: Phuong
authors.initials: P
authors.lastname: Nguyen
authors.affiliation: 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.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Wan
authors.affiliation: Departments of Neurosciences and Bioengineering, University of California, 9500 Gilman Drive La Jolla, San Diego, CA 92093-0608, USA.|Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Lanman
authors.affiliation: 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.
authors.forename: Albert
authors.initials: A
authors.lastname: Lawrence
authors.affiliation: 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.|Departments of Neurosciences and Bioengineering, University of California, 9500 Gilman Drive La Jolla, San Diego, CA 92093-0608, USA.|Department of Biological Sciences, Purdue University, 915 W. State Street, West Lafayette, IN 47907-2054 USA.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
keywords: 3D reconstruction; Electron tomography; Iterative methods; Tomogram averaging; TxBR