Abstract
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.
Highlights
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]
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 200–500nm thick samples [2] eliminating the need for finer sectioning to visualize the sample volume
Summary
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]. 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 1–2u) 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. 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]
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