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Three-dimensional electron microscopy of entire cells.

Swiss National Centre for Retroviruses, Zürich, Switzerland.
Journal of Microscopy (Impact Factor: 2.15). 02/1990; 157(Pt 1):115-26. DOI: 10.1111/j.1365-2818.1990.tb02952.x
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ABSTRACT The digital processing of serial electron-microscope sections containing laser-induced topographical references allows a three-dimensional (3-D) reconstruction of entire cells at a depth resolution of 40-60 nm by the use of novel image analysis methods. The images are directly processed by a video-camera placed under the electron microscope in TEM mode or by the electron counting device in STEM mode. The deformations associated with the cutting of embedded cells are back-calculated by new computer algorithms developed for image analysis and treatment. They correct the artefacts caused by serial sectioning and automatically reconstruct the third dimension of the cells. Used in such a way, our data provide definitive information on the 3-D architecture of cells. This computer-assisted 3-D analysis represents a new tool for the documentation and analysis of cell ultrastructure and for morphometric studies. Furthermore, it is now possible for the observer to view the contents of the reconstructed tissue volume in a variety of different ways using computer-aided display techniques.

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    • "A few years later Glaser and Glaser (1965) demonstrated a computer generated 3D-reconstruction from images derived from serial sectioning analyzed by a light microscope. Since these striking works many articles dealing with different kinds of 3D-reconstructions based on microscopical data (transmission electron microscope (Bron et al., 1990; Gremillet et al., 1991), lightmicroscopy of semithin-sections (Schmolke and Fleischhauer, 1984; Schmolke, 1996), lightmicroscopy of 5 to 50 μm thick sections have been published (Schormann and Zilles, 1998; Ourselin et al., 2001b). A disadvantage of sectioning, staining and mounting the sections on the slides is a lack of spatially perfect matching when the sections are superimposed. "
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    ABSTRACT: The physical (microtomy), optical (microscopy), and radiologic (tomography) sectioning of biological objects and their digitization lead to stacks of images. Due to the sectioning process and disturbances, movement of objects during imaging for example, adjacent images of the image stack are not optimally aligned to each other. Such mismatches have to be corrected automatically by suitable registration methods. Here, a whole brain of a Sprague Dawley rat was serially sectioned and stained followed by digitizing the 20 μm thin histologic sections. We describe how to prepare the images for subsequent automatic intensity based registration. Different registration schemes are presented and their results compared to each other from an anatomical and mathematical perspective. In the first part we concentrate on rigid and affine linear methods and deal only with linear mismatches of the images. Digitized images of stained histologic sections often ex- hibit inhomogenities of the gray level distribution coming from staining and/or sectioning variations. Therefore, a method is developed that is robust with respect to inhomogenities and artifacts. Furthermore we combined this approach by minimizing a suitable distance measure for shear and rotation mismatches of foreground ob- jects after applying the principal axes transform. As a consequence of our investigations, we must emphasize that the combination of a robust principal axes based registration in combination with optimizing translation, rota- tion and shearing errors gives rise to the best reconstruction results from the mathematical and anatomical view point. Because the sectioning process introduces nonlinear deformations to the relative thin histologic sections as well, an elastic registration has to be applied to correct these deformations. In the second part of the study a detailed description of the advances of an elastic registration after affine linear registration of the rat brain is given. We found quantitative evidence that affine linear registration is a suitable starting point for the alignment of histologic sections but elastic registration must be performed to improve significantly the registration result. A strategy is presented that enables to register elastically the affine linear preregistered rat brain
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    • "Elucidation of the relative positioning, shape and size of plant parenchyma cells in situ has hitherto been achieved by indirect methods involving microscopic analyses of thin, serial sections followed by stereological analysis (Considine, 1978; Considine, 1981; Cruz- Orive, 1997; Mùller et al., 1990; Underwood, 1970; Weibel, 1979; Weibel, 1980) or three-dimensional (3D) reconstruction of physical slices (Bron et al., 1990; Korn and Spalding, 1973; Korn, 1974; Lewis, 1926; Matzke, 1948; Williams, 1968). While appropriate for tissues composed of isodiametric cells, stereological methods underestimate mean cell sizes (Considine and Knox, 1981) and do not allow accurate determinations of intratissue variation (Parsons et al., 1989). "
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    The Plant Journal 08/1999; 19(2):229-236.
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    • "Seventy nanometer-thick ultrathin sections were cut with a Ultracut S ultramicrotome (Reichert-Jung) equipped with a diamond knife (Diatome). Sections were submitted to chloroform vapor to correct possible deformations due to compression (Bron et al, 1990). Sections were collected with 150 mesh copper grids, stained with 2% uranyl acetate in 50% ethanol, and poststained with 0.2% lead citrate. "
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