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Choice of section thickness in serial-section electron microscopy
JOURNAL OF ULTRASTRUCTURE RESEARCH
88,
287-311
(1984)
Abstracts (Biology) from the Thirty-Seventh Annual Meeting of the Scandinavian Society for Electron Microscopy The annual meeting of the Scandinavian Society for Electron Microscopy. "SCANDEM-84," was held in Copenhagen, Denmark, June 6-8, 1984. Given below are titles and abstracts of the biological papers presented there.
Ultracytochemical Localization of/~-Galactosidase Activity in Peritoneal Macrophages from C57B1 Mice. J. BLOM AND J. M. RHODES, Department of Biophysics and International Escherichia and Klebsiella Centre, Statens Seruminstitut, DK-2300 Copenhagen S, Denmark. A cytochemical method for the detection of the acid hydrolase fl-galactosidase (fl-gal) by light microscopy was adapted for electron microscopy. Using this method it was found that the reaction product for fl-gal (GRP) in peritoneal macrophages (PMO) from untreated C57B1 mice was localized in the perinuclear cisternae, the RER, the Golgi complex, lysosomes, vesicles, and on the cell surface. PMO were heterogeneous with respect to their content of ~-gal, but direct exposure to an inflammatory stimulus by injection of Propionibacterium acne ip caused a visible loss in the fl-gal content of PMO. The sites in which fl-gal is situated intracellularly suggest that this enzyme may be easily secreted by exocytosis, and this may partly explain why there is a loss in activity after direct exposure to P. acne. When examined by X-ray microanalysis, the crystalline GRp was found to contain bromine, an element which is present in the fl-gal indolyl substrate. The bromine was only detected in the crystals and not in the adjacent cytoplasm. A further point of interest from the X-ray microanalysis was the finding of G RP crystals on the outer membrane of some erythrocytes, in eosinophilic leukocytes, and occasionally in lymphocytes. X-Ray microanalysis is thus a valuable tool for the analysis of reaction products when substrates containing unphysiological elements such as bromine are used.
Choice of Section Thickness in Serial-Section Electron Microscopy. M. BUNDGAARD, Department of Medical Physiology A, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Three-dimensional reconstructions based on consecutive thin sections are often required in order to reveal the true shape of cellular structures. The thickness of the consecutive sections determines the resolution of the reconstructions. The thinner the sections, the more detailed the reconstructions can be made. Therefore the thickness must be adjusted to the dimensions of structures under investigation. However, it is not possible to calculate precisely how thin the sections need to be. As a rule of thumb, the sections must be thinner than the dimension of the smallest structural detail of interest. The required thickness has to be specified by trial and error during electron microscopy of short ribbons of various thickness. The importance of section thickness for the outcome of threedimensional reconstructions can be illustrated by studies of the organization of the characteristic vesicular profiles in capillary endothelia. Reconstructions based on 50-nm sections indicated that free plasmalemmal vesicles often occur in the cytoplasm. However, reconstructions based on consecutive sections, less than 15 nm thick, showed that the vesicular profiles do not represent true vesicles. The profiles turned out to be parts of more or less complex invaginations of the plasmalemma. In the series of 50-nm sections 287 0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
288
SCANDEM-84
the continuity between vesicular profiles was often lost. The ultrathin sections allowed all "vesicle-vesicle" and "vesicle-plasmalemma" fusions to be identified (M. Bundgaard, 1983, Fed. Proc. 42, 2425-2430).
Autometalography. G. DANSCHERAND J. O. RYTTER NORGAARD, Institute of Anatomy,
University of Aarhus, Denmark. A new method is described for the autoinduced silver amplification of certain metal atoms and molecules in sections of biological specimens at both light and electron microscopic level. In the autometalographic (AM) technique a commercial photographic emulsion is placed on slides or grids which are subsequently exposed to a weak alkaline chemical developer. The catalytic metal located in the tissue will induce a reduction of silver ions originating from the silver bromide crystals of the emulsion and as a result the catalysts will be encapsulated in spheres of metalic silver. The method is applicable for the visualization of gold, silver, metal selenids, and sulfides. An an example of AM at the light microscopic level, cryostat sections from rat brains perfused with sulfide are presented. A delicate pattern of silver-amplified zinc sulfide with a nonspecific background is observed. At the ultrastructural level AM is performed on sections from rats treated with aurothiomalatate, silver lactate, and mercury chloride. In addition, the time-dependent silver amplification of AM is demonstrated using particles of colloidal gold dispersed in a film of gelatin. In the tissue sections otherwise invisible accumulations of metal catalyst are clearly seen as particulate silver grains. Unspecific grains caused by background radiation can easily be distinguished in the EM because of their ball-of-yarn appearance. Colloidal gold particles could be enlarged 3.5 times in diameter after 15 min of development corresponding to a 12-fold increase in area and are consequently easily located at low magnifications. AM has proved to be a fast, reliable, and specific method for demonstration of gold, silver, metal sulfides, and selenides in biological specimens.
Some Analytical Capabilities of a High-Resolution STEM and Their Application in Biology. A. ENGELAND R. REICHELT,Department of Microbiology, Biozentrum, University
of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. A scanning transmission electron microscope (STEM) allows a variety of signals to be collected simultaneously and displayed as images. Due to sequential illumination of small sample volumes (typically 1 x 1 x 10 nm3), these signals can be digitized on-line and transferred to a computer. Thus, multiple high-resolution STEM images are directly available for quantitative evaluation and permit analytical studies to be done with great efficiency. Using the annular detector signal, which represents approximately 70% of all elastically scattered electrons, the mass of single proteins and their supramolecular assemblies is determined with attractive resolution. This is interesting for the biochemist, since mass analysis can be accomplished from a small amount of material in aggregated as well as solubilized form. Combining then the elastic and inelastic signals (the latter collected by a/J-spectrometer) as normalized ratio, the local and integral concentration of biological matter within a thin section is measurable. Preliminary results show, e.g., for protein embedded in Lowicryl HM20 that the full concentration range (0-100%) can be subdivided into five to nine concentration intervals, if sample volumes of typically 103-104 nm 3 are investigated. Element mapping exploits the information carried by the electron energy-loss spectrum and allows the depiction of the specific element distribution at moderate resolution. Poor signal-to-noise ratio and the problem of appropriate back-
88,
287-311
(1984)
Abstracts (Biology) from the Thirty-Seventh Annual Meeting of the Scandinavian Society for Electron Microscopy The annual meeting of the Scandinavian Society for Electron Microscopy. "SCANDEM-84," was held in Copenhagen, Denmark, June 6-8, 1984. Given below are titles and abstracts of the biological papers presented there.
Ultracytochemical Localization of/~-Galactosidase Activity in Peritoneal Macrophages from C57B1 Mice. J. BLOM AND J. M. RHODES, Department of Biophysics and International Escherichia and Klebsiella Centre, Statens Seruminstitut, DK-2300 Copenhagen S, Denmark. A cytochemical method for the detection of the acid hydrolase fl-galactosidase (fl-gal) by light microscopy was adapted for electron microscopy. Using this method it was found that the reaction product for fl-gal (GRP) in peritoneal macrophages (PMO) from untreated C57B1 mice was localized in the perinuclear cisternae, the RER, the Golgi complex, lysosomes, vesicles, and on the cell surface. PMO were heterogeneous with respect to their content of ~-gal, but direct exposure to an inflammatory stimulus by injection of Propionibacterium acne ip caused a visible loss in the fl-gal content of PMO. The sites in which fl-gal is situated intracellularly suggest that this enzyme may be easily secreted by exocytosis, and this may partly explain why there is a loss in activity after direct exposure to P. acne. When examined by X-ray microanalysis, the crystalline GRp was found to contain bromine, an element which is present in the fl-gal indolyl substrate. The bromine was only detected in the crystals and not in the adjacent cytoplasm. A further point of interest from the X-ray microanalysis was the finding of G RP crystals on the outer membrane of some erythrocytes, in eosinophilic leukocytes, and occasionally in lymphocytes. X-Ray microanalysis is thus a valuable tool for the analysis of reaction products when substrates containing unphysiological elements such as bromine are used.
Choice of Section Thickness in Serial-Section Electron Microscopy. M. BUNDGAARD, Department of Medical Physiology A, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark. Three-dimensional reconstructions based on consecutive thin sections are often required in order to reveal the true shape of cellular structures. The thickness of the consecutive sections determines the resolution of the reconstructions. The thinner the sections, the more detailed the reconstructions can be made. Therefore the thickness must be adjusted to the dimensions of structures under investigation. However, it is not possible to calculate precisely how thin the sections need to be. As a rule of thumb, the sections must be thinner than the dimension of the smallest structural detail of interest. The required thickness has to be specified by trial and error during electron microscopy of short ribbons of various thickness. The importance of section thickness for the outcome of threedimensional reconstructions can be illustrated by studies of the organization of the characteristic vesicular profiles in capillary endothelia. Reconstructions based on 50-nm sections indicated that free plasmalemmal vesicles often occur in the cytoplasm. However, reconstructions based on consecutive sections, less than 15 nm thick, showed that the vesicular profiles do not represent true vesicles. The profiles turned out to be parts of more or less complex invaginations of the plasmalemma. In the series of 50-nm sections 287 0022-5320/84 $3.00 Copyright © 1984 by Academic Press, Inc. All rights of reproduction in any form reserved.
288
SCANDEM-84
the continuity between vesicular profiles was often lost. The ultrathin sections allowed all "vesicle-vesicle" and "vesicle-plasmalemma" fusions to be identified (M. Bundgaard, 1983, Fed. Proc. 42, 2425-2430).
Autometalography. G. DANSCHERAND J. O. RYTTER NORGAARD, Institute of Anatomy,
University of Aarhus, Denmark. A new method is described for the autoinduced silver amplification of certain metal atoms and molecules in sections of biological specimens at both light and electron microscopic level. In the autometalographic (AM) technique a commercial photographic emulsion is placed on slides or grids which are subsequently exposed to a weak alkaline chemical developer. The catalytic metal located in the tissue will induce a reduction of silver ions originating from the silver bromide crystals of the emulsion and as a result the catalysts will be encapsulated in spheres of metalic silver. The method is applicable for the visualization of gold, silver, metal selenids, and sulfides. An an example of AM at the light microscopic level, cryostat sections from rat brains perfused with sulfide are presented. A delicate pattern of silver-amplified zinc sulfide with a nonspecific background is observed. At the ultrastructural level AM is performed on sections from rats treated with aurothiomalatate, silver lactate, and mercury chloride. In addition, the time-dependent silver amplification of AM is demonstrated using particles of colloidal gold dispersed in a film of gelatin. In the tissue sections otherwise invisible accumulations of metal catalyst are clearly seen as particulate silver grains. Unspecific grains caused by background radiation can easily be distinguished in the EM because of their ball-of-yarn appearance. Colloidal gold particles could be enlarged 3.5 times in diameter after 15 min of development corresponding to a 12-fold increase in area and are consequently easily located at low magnifications. AM has proved to be a fast, reliable, and specific method for demonstration of gold, silver, metal sulfides, and selenides in biological specimens.
Some Analytical Capabilities of a High-Resolution STEM and Their Application in Biology. A. ENGELAND R. REICHELT,Department of Microbiology, Biozentrum, University
of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. A scanning transmission electron microscope (STEM) allows a variety of signals to be collected simultaneously and displayed as images. Due to sequential illumination of small sample volumes (typically 1 x 1 x 10 nm3), these signals can be digitized on-line and transferred to a computer. Thus, multiple high-resolution STEM images are directly available for quantitative evaluation and permit analytical studies to be done with great efficiency. Using the annular detector signal, which represents approximately 70% of all elastically scattered electrons, the mass of single proteins and their supramolecular assemblies is determined with attractive resolution. This is interesting for the biochemist, since mass analysis can be accomplished from a small amount of material in aggregated as well as solubilized form. Combining then the elastic and inelastic signals (the latter collected by a/J-spectrometer) as normalized ratio, the local and integral concentration of biological matter within a thin section is measurable. Preliminary results show, e.g., for protein embedded in Lowicryl HM20 that the full concentration range (0-100%) can be subdivided into five to nine concentration intervals, if sample volumes of typically 103-104 nm 3 are investigated. Element mapping exploits the information carried by the electron energy-loss spectrum and allows the depiction of the specific element distribution at moderate resolution. Poor signal-to-noise ratio and the problem of appropriate back-