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
Source: PubMed

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.

Download full-text


Available from: Michel Jourlin, Oct 22, 2014
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
    International Journal of Computer Vision 01/2007; 73:5-39. DOI:10.1007/s11263-006-9780-x
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Parenchyma cells from the inner mesocarp of a grape berry (Vitis vinifera L. cv. Chardonnay) were visualised in three-dimensions within a whole mount of cleared, stained tissue using confocal laser scanning microscopy and digital image reconstruction. The whole berry was fixed, bisected longitudinally, cleared in methyl salicylate, stained with safranin O and mounted in methyl salicylate. Optical slices were collected at 1.0 µm intervals to a depth of 150 µm. Neighbouring z-series were joined post-collection to double the field-of-view. Attenuation at depth of the fluorescent signal from cell walls was quantified and corrected. Axial distortion due to refractive index mismatch between the immersion and mounting media was calibrated using yellow-green fluorescent microspheres and corrected. Transmission electron microscopy was used to correct fluorescent measurements of cell wall thickness. Digital image reconstructions of wall-enclosed spaces enabled cells to be rendered as geometric solids of measurable surface area and volume. Cell volumes within the inner mesocarp tissue of a single grape berry exhibited a 14-fold range, with polysigmoidal distribution and groupings around specific size classes. Cell shape was irregular and the planes of contact were rarely flat or simple. Variability in cell shape was indicated by the range in surface area to volume ratios, from 0.080 to 0.198 µm-1. Structural detail at the internal surface of the cell wall was apparent. The technique is applicable to a wide range of morphometric analyses in plant cell biology, particularly developmental studies, and reveals details of cell size and shape that were previously unattainable.
    The Plant Journal 08/1999; 19(2):229-236.
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The aim of the study was to compare two methods of quantitating eumelanins and pheomelanins, pigments synthesized by melanocytes. One is based on the high performance liquid chromatography quantitation of specific degradation products of each melanin type. The other requires image analysis, transmission electron microscopy, and stereology. In a previous study, we showed good correlations between both methods for total melanin but not for eumelanins or pheomelanins. We describe here the same comparison in more pigmented cells (nevus cells and stimulated HBL melanoma cells). Transmission electron microscopy micrographs were image analyzed to generate several primary parameters. Stereology was used for estimating melanosomal maturation, intracellular melanin content, and the number of melanized melanosomes per cell, for total melanin, eumelanins, or pheomelanins. Our results showed a good correlation between both methods for total melanin, eumelanins, and pheomelanins with an r equal to 099, 0.91, and 0.93, respectively, when all the points were used in the linear regression analyses. In the melanoma cell group (HBL cells cultured in media of different compositions), the chemical and morphometric estimations were not parallel in the case of eumelanins and pheomelanins. In addition, the stereologic and high performance liquid chromatography pheomelanins to eumelanins ratios were still not correlated. These results demonstrate the relevancy of the stereologic method, but the low level of melanization, the possible lack of specificity of melanogenesis in melanoma cells, and a problem of sensitivity of the stereologic method in this context seem to be obstacles in obtaining better results. The utilization of normal human melanocytes could give some answers to our hypotheses.Keywords: alkali elution, morphometry, normal epidermal melanocytes, transmission electron microscopy
    Journal of Investigative Dermatology 08/1998; 111(3):422-428. DOI:10.1046/j.1523-1747.1998.00313.x
Show more