ISSN:
0741-0581
Keywords:
Serial sections
;
3D reconstruction
;
Image analysis
;
HRSEM
;
TEM
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Natural Sciences in General
Notes:
Following stereo, high resolution scanning electron microscopy (HRSEM) of various rat tissue mitochondria prepared by mild glutaraldehyde fixation followed by freeze cleavage and extraction of the cytosol (Lea and Hollenberg, 1989a), a new model for the ultrastructure of mitochondrial cristae has been developed (Lea and Hollenberg, 1989b). The cristae mitochondriales, previously considered in most cells to have a shelf like structure, were found in many cell types including hepatocytes by HRSEM to be tubes which spanned the mitochondrial matrix and were continuous with the inner mitochondrial membrane at both ends. Despite computer aided, serial, reconstruction of serial thin sections examined in the transmission electron microscope (TEM), tubular cristae in mitochondria have not been resolved entirely, even though circular cross sectional profiles of cristae have been observed in the TEM. The current study was undertaken to probe this deficiency and to understand its cause.In this study, the problems of inherent contrast and resolution loss in the digitized image have been reduced by using a computer based, background, extraction process analogous in the computer to the chemical cytosol extraction process used for HRSEM (Lea and Hollenberg, 1988; Hollenberg et al., 1989). Despite the use of this membrane enhancement algorithm, it was still not possible to reconstruct entire tubular cristae, as observed by HRSEM, even when the micrograph negatives were digitized and reprocessed (Lea and Hollenberg, 1989b). A comparison of serial section thickness deduced from the diffraction colour of the respective section (60 nm) to the measured diameter of tubular cristae profiles (30 nm), with membrane thickness of 10 nm, suggested that this size disparity is responsible for the apparent loss of resolution. The sampling theorem from signal analysis indicates that the signals of interest (X, Y vs. Z coordinate values of mitochondrial internal structure) must be sampled at least twice the frequency of their highest frequency. Because the structures are small and embedded in image noise, even thinner serial sections are required in order to obtain enough information for adequate reconstruction. Consequently in practice, the thickness of the serial sections would have to be reduced by four times to less than 8 nm in order to be able to reconstruct tubular mitochondrial cristae 30 nm in diameter and less than 2 nm for membrane shape. Ultra-thin sections, some 15 nm in thickness, have been reported (Buchanan et al., 1988). However, these sections would be only thin enough to resolve structures greater than 30 nm in diameter. Observation of cristae by HRSEM therefore is essential in order to verify ultrastructure derived by computer based three dimensional reconstruction.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jemt.1060180306
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