Image processing and 3-D reconstruction in electron microscopy (original) (raw)
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Journal of Synchrotron Radiation, 2004
Although the methodology of molecular microscopy has enormous potential, it is time consuming and labor intensive. The techniques required to produce a three dimensional (3D) electron density map of a macromolecular structure normally require manual operation of an electron microscope by a skilled operator and manual supervision of the sometimes complex software needed for analysis and calculation of 3D maps. We are developing systems to automate the process of data acquisition from an electron microscope and integrating these systems with specimen handling operations and post acquisition data processing. We report here on the current performance of our existing systems and the future challenges involved in substantially improving both the sustained throughput and the yield of automated data collection and analysis.
THREE DIMENSIONAL ELECTRON MICROSCOPY AND IN SILICO TOOLS FOR MACROMOLECULAR STRUCTURE DETERMINATION
Recently, structural biology witnessed a major tool -electron microscopy -in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it.
Protein structure determination by electron cryo-microscopy
Current Opinion in Pharmacology, 2009
Transmission electron cryo-microscopy (cryoEM) is a versatile tool in the structural analysis of proteins and biological macromolecular assemblies. In this review, we present a brief survey of the methods used in cryoEM, and their current developments. These latest advances provide exciting opportunities for the three-dimensional structural determination of macromolecular complexes that are either too large or too heterogeneous to be investigated by conventional X-ray crystallography or nuclear magnetic resonance (NMR). The endeavour of understanding the function of protein or macromolecular complex is often helped by combining data from electron microscopy and X-ray crystallography. We will thus provide a brief overview of the computational techniques involved in combining data from different techniques for the interpretation of the EM structure.
The Emergence of Electron Tomography as an Important Tool for Investigating Cellular Ultrastructure
Journal of Histochemistry & Cytochemistry, 2001
Electron tomography has emerged as the leading method for the study of three-dimensional (3D) ultrastructure in the 5–20-nm resolution range. It is ideally suited for studying cell organelles, subcellular assemblies and, in some cases, whole cells. Tomography occupies a place in 3D biological electron microscopy between the work now being done at near-atomic resolution on isolated macromolecules or 2D protein arrays and traditional serial-section reconstructions of whole cells and tissue specimens. Tomography complements serial-section reconstruction by providing higher resolution in the depth dimension, whereas serial-section reconstruction is better able to trace continuity over long distances throughout the depth of a cell. The two techniques can be combined with good results for favorable specimens. Tomography also complements 3D macromolecular studies by offering sufficient resolution to locate the macromolecular complexes in their cellular context. The technology has matured t...
Microscopy (Oxford, England), 2015
Single particle cryo-EM has recently developed into a powerful tool to determine the 3D structure of macromolecular complexes at near-atomic resolution, which allows structural biologists to build atomic models of proteins. All technical aspects of cryo-EM technology have been considerably improved over the last two decades, including electron microscopic hardware, image processing software and the ever growing speed of computers. This leads to a more widespread use of the technique, and it can be anticipated that further automation of electron microscopes and image processing tools will soon fully shift the focus away from the technological aspects, onto biological questions that can be answered. In single particle cryo-EM, no crystals of a macromolecule are required. In contrast to X-ray crystallography, this significantly facilitates structure determination by cryo-EM. Nevertheless, a relatively high level of biochemical control is still essential to obtain high-resolution struct...
Electron Tomography of Large, Multicomponent Biological Structures
Journal of Structural Biology, 1997
Electron tomography is an extremely useful method for deriving three-dimensional structure from electron microscope images. The application of this technique to the reconstruction of large, complex structures such as mitochondria is described in conjunction with several tools for segmentation, measurement, classification, and visualization. In addition, the use of massively parallel computers to perform the tomographic reconstruction efficiently using R-weighted backprojection or iterative techniques is described. 1997 Academic Press
Perspectives of Molecular and Cellular Electron Tomography
Journal of Structural Biology, 1997
After a general introduction to three-dimensional electron microscopy and particularly to electron tomography (ET), the perspectives of applying ET to native (frozen-hydrated) cellular structures are discussed. In ET, a set of 2-D images of an object is recorded at different viewing directions and is then used for calculating a 3-D image. ET at a resolution of 2-5 nm would allow the 3-D organization of structural cellular components to be studied and would provide important information about spatial relationships and interactions. The question of whether it is a realistic long-term goal to visualize or-by sophisticated pattern recognition methodsidentify macromolecules in cells frozen in toto or in frozen sections of cells is addressed. Because of the radiation sensitivity of biological specimens, a prerequisite of application of ET is the automation of the imaging process. Technical aspects of automated ET as realized in Martinsried and experiences are preresented, and limitations of the technique are identified, both theoretically and experimentally. Possible improvements of instrumentation to overcome at least part of the limitations are discussed in some detail. Those means include increasing the accelerating voltage into the intermediate voltage range (300 to 500 kV), energy filtering, the use of a field emission gun, and a liquid-helium-cooled specimen stage. Two additional sections deal with ET of isolated macromolecules and of macromolecular structures in situ, and one section is devoted to possible methods for the detection of structures in volume data. 1997 Academic Press
Three-dimensional electron cryo-microscopy as a powerful structural tool in molecular medicine
Electron cryo-microscopy has established itself as a valuable method for the structure determination of protein molecules, protein complexes, and cell organelles. This contribution presents an introduction to the various aspects of three-dimensional electron cryomicroscopy. This includes the need for sample preservation in the microscope vacuum, strategies for minimizing radiation damage, methods of improving the poor signalto-noise ratio in electron micrographs of unstained specimens, and the various methods of three-dimensional image reconstruction from projections. The various specimen types (e.g., flat and tubular two-dimensional crystals, protein filaments, individual protein molecules, and large complexes) require different means of three-dimensional reconstruction, and we review the five major reconstruction techniques (electron crystallography, heli-cal reconstruction, icosahedral reconstruction, singleparticle reconstruction, and electron tomography), with an emphasis on electron crystallography. Several medically relevant three-dimensional protein structures are chosen to illustrate the potential of electron cryo-microscopy and image reconstruction techniques. Among the structural methods, electron cryo-microscopy is the only tool for studying objects that range in size from small proteins over macromolecular complexes to cell organelles or even cells.
Electron tomography of membrane-bound cellular organelles
2006
Electron microscope tomography produces three-dimensional reconstructions and has been used to image organelles both isolated and in situ, providing new insight into their structure and function. It is analogous to the various tomographies used in medical imaging. Compared with light microscopy, electron tomography offers an improvement in resolution of 30-to 80-fold and currently ranges from 3 to 8 nm, thus filling the gap between high-resolution structure determinations of isolated macromolecules and larger-scale studies on cells and tissues by light microscopy. Here, we provide an introduction to electron tomography and applications of the method in characterizing organelle architecture that also show its power for suggesting functional significance. Further improvements in labeling modalities, imaging tools, specimen preparation, and reconstruction algorithms promise to increase the quality and breadth of reconstructions by electron tomography and eventually to allow the mapping of the cellular proteomes onto detailed three-dimensional models of cellular structure. TEM: transmission electron microscopy Electron tomography (ET): the process of calculating the three-dimensional structure of a specimen from a tilt series of electron micrographs representing two-dimensional projections of the three-dimensional structure Contents
Electron tomography: A 3d view of the subcellular world
E lectron microscopy (EM) revolutionized cell biology in the 1960s when it revealed details of cellular ultrastructure. Sections of cells were preserved well enough and cut thin enough to allow examination with the electron microscope at a resolution ~100× better than was possible by light microscopy. With improvements in light microscopes and the advent of genetically encoded fluorescent proteins during the past 20 years, however, EM has taken a backseat as fluorescence microscopy became the dominant method for studying cellular processes; this was due in part to light microscopy's ability to image the dynamic behavior of proteins in live cells. A convergence of technological advances has enabled biological applications of electron tomography, thereby expanding the range of accessible size scales and complexity. © 2 0 0 7 A m e r i c A n c h e m i c A l S o c i e t y n o v e m b e r 1 , 2 0 0 7 / A n A l y t i c A l c h e m i S t r y 7 9 4 9