Compensation of Missing Wedge Effects with Sequential Statistical Reconstruction in Electron Tomography

et al. (2014) Compensation of Missing Wedge Effects with Sequential Statistical Reconstruction in Electron Tomography. PLoS ONE 9(10): e108978. doi:10.1371/journal.pone.0108978 Compensation of Missing Wedge Effects with Sequential Statistical Reconstruction in Electron Tomography Lassi Paavolainen 0 Erman Acar 0 Uygar Tuna 0 Sari Peltonen 0 Toshio Moriya 0 Pan Soonsawad 0 Varpu Marjoma ki 0 R. Holland Cheng 0 Ulla Ruotsalainen 0 Eve-Isabelle Pecheur, UMR Inserm U1052/CNRS 5286, France 0 1 Department of Biological and Environmental Science/Nanoscience Center, University of Jyva skyla , Jyva skyla , Finland, 2 Department of Signal Processing, Tampere University of Technology , Tampere, Finland, 3 BioMediTech , Tampere University of Technology , Tampere , Finland , 4 Department of Molecular and Cellular Biology, University of California Davis, Davis, California, United States of America, 5 Department of Anatomy, Faculty of Dentistry, Mahidol University , Bangkok , Thailand Electron tomography (ET) of biological samples is used to study the organization and the structure of the whole cell and subcellular complexes in great detail. However, projections cannot be acquired over full tilt angle range with biological samples in electron microscopy. ET image reconstruction can be considered an ill-posed problem because of this missing information. This results in artifacts, seen as the loss of three-dimensional (3D) resolution in the reconstructed images. The goal of this study was to achieve isotropic resolution with a statistical reconstruction method, sequential maximum a posteriori expectation maximization (sMAP-EM), using no prior morphological knowledge about the specimen. The missing wedge effects on sMAP-EM were examined with a synthetic cell phantom to assess the effects of noise. An experimental dataset of a multivesicular body was evaluated with a number of gold particles. An ellipsoid fitting based method was developed to realize the quantitative measures elongation and contrast in an automated, objective, and reliable way. The method statistically evaluates the sub-volumes containing gold particles randomly located in various parts of the whole volume, thus giving information about the robustness of the volume reconstruction. The quantitative results were also compared with reconstructions made with widely-used weighted backprojection and simultaneous iterative reconstruction technique methods. The results showed that the proposed sMAP-EM method significantly suppresses the effects of the missing information producing isotropic resolution. Furthermore, this method improves the contrast ratio, enhancing the applicability of further automatic and semi-automatic analysis. These improvements in ET reconstruction by sMAP-EM enable analysis of subcellular structures with higher three-dimensional resolution and contrast than conventional methods. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: Funding provided by National Institute of Health (AI095382) http://www.nih.gov/, National Institute of Food and Agriculture http://www.csrees.usda. gov/, Tekes FiDiPro, (1913/31/2012) http://www.tekes.fi/en/fidipro/, Discovery Grant (UCDG178969) and Tampere University of Technology Internal Grant http:// www.tut.fi/en/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Electron tomography (ET) of cellular samples is a widely used technique for three-dimensional (3D) reconstruction of complex subcellular structures at a resolution enabling the detection of macromolecular complex organizations [1]. Generally, in tomography a 3D model of an object is reconstructed from a collection of two-dimensional (2D) projection images of the sample taken in multiple orientations. In its simplest form, either the sample or the radiation source and detector are rotated around a single axis for full 180 or 360 degrees with fixed intervals (typically 12u) while projection images are taken [1]. ET is a combination of this computed tomography and electron microscopy, and fills the resolution gap between the structural methods at the subnanometer level, such as single-particle reconstruction, and those at the sub-micrometer level using optical microscopy. In ET, transmission electron microscope (TEM) is used to image typically 200500nm thick samples [2] eliminating the need for finer sectioning to visualize the sample volume. Using modern sample preparation techniques, like cryo-electron microscopy methods, ET enables studying the physiological mechanisms of subcellular organelles in their native context [1,2]. To understand the highly complex mechanisms of cell activities, like the signal pathways and mechanisms of virus infections, it is crucial to study various aspects of the cell such as the morphological abnormalities of the whole cell, organelles, or intracellular compartment membranes, and localization or distribution changes of related proteins. These phenomena occur in 3D space. However, while the TEM imaging allows a high resolution observation, the images lose the information of the density distribution along the z-direction (direction of the electron beam, depth), occluding the fine features and distance between two objects along this direction. Therefore, the 3D reconstructions of the target objects using ET have been important for these biological studies. However, ET of biological samples holds two major limitations in image acquisition of tilt series for tomography: a limited range of observable tilt angles and an extremely low signal-to-noise ratio (SNR). In the general case, the sample cannot be imaged in full 180u tilt angle range since the structure of the sample holder and limited space between the pole pieces of the objective lens prevent acquiring images with high tilt angles [3]. This missing angular range is known as missing wedge. In addition, at 60u tilt angle, the electron beam has to pass approximately twice as much material of the slab-shaped sample as at zero degree [4]. This makes the quality of high tilt angle images worse. Typically, 660u70u tilt angle range is used in ET. Together, these technological challenges cause anisotropic resolution seen as elongation and blurring of the objects in the z-direction. In order to reduce the missing information and minimize these artifacts, additional data is acquired from different tilt axes (double tilt axis [5,6], conical tilt axis [7]). The missing wedge is reduced to a missing pyramid with double tilt axis, and a missing cone with conical tilt axis by increasing the information coverage in the Fourier domain. Nevertheless, the usage of these techniques has no (...truncated)