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Electron microscopy of cells: a new beginning for a new century.

McIntosh JR - J. Cell Biol. (2001)

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA. richard.mcintosh@colorado.edu

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Thus, in spite of its value for antigen localization, the approach is not an ideal way to study cell morphology... Its reliance on both chemical fixation and infiltration with an osmotically active cryoprotectant reduces the likelihood that the resulting images will reflect the condition in a living cell... Finally, there are indications that an electron energy loss filter, developed by several companies to remove electrons that were inelastically scattered by the specimen before an image is formed, can enhance the quality of images from thick, frozen-hydrated specimens (Grimm et al. 1997)... Although these technologies have led to significant progress in imaging frozen cells, there are still formidable problems to overcome before this approach will yield the most useful results... These problems have recently been addressed by combining the power of RFFSE with tomography to obtain 3-D views of cellular structures that should closely resemble the living state... The accuracy of these specimen preparation methods has been tested in three ways: (a) significant fractions (1/20–1/5) of rapidly frozen yeast cells are viable upon rewarming, showing that rapid freezing itself preserves the native structure of cells very well. (b) RFFSE samples have been compared with equivalent ones prepared by freeze-fracture EM, a method that visualizes replicas of fractured cellular surfaces formed by metal shadowing at very low temperatures... These studies have found no significant difference between the structures seen by the two methods (e.g., Linder and Staehelin 1979). (c) The images from samples prepared by RFFSE conform to expectations that are based on a broad range of knowledge about subcellular components: cytoskeletal fibers usually run straight; membrane profiles of the ER and of Golgi cisternae appear turgid and smooth; most vesicles are round; cytomatrix is even; even chromatin appears structured (McDonald and Morphew 1993; Ladinsky et al. 1999; Muller et al. 2000)... Thus, studies that have melded RFFSE with tomography are likely to be showing us cellular structures that are essentially native, revealing organelle morphology in its normal context and in a situation where the parameters of cell physiology, like cell cycle stage, can be manipulated by the experimenter... An additional advantage of EM tomography is that many cellular structures are visualized at once with a single imaging technology... EM tomography of well-fixed cells, on the other hand, reveals the relationships among diverse cellular structures at comparatively high resolution (Fig. 5)... The tomograms of RFFSE samples already show enough detail and quality to prompt serious study in their own right, and it is realistic to think about using these methods to view major cellular subsystems, like an entire nucleus or a Golgi complex... Of more concern is the issue of recognizing macromolecules of interest within cellular tomograms... We see a brave new world emerging from the combination of modern EM with the rapidly advancing methods for light microscopy... The resulting data should help us to understand the context for functional genomics and elucidate the continuum of structural order that bridges from inanimate molecules to the order of the living cell.

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Stereo views of a model generated by tomographic reconstruction of a slab of cytoplasm from a cultured cell, strain HIT-T15. Three serial 400-nm sections were reconstructed by dual axis tomography, and the IMOD software package was used to model all visible objects within a volume 3.1 × 3.2 × 1.2 μm3 (see Marsh et al. 2001 for details). The model displays the Golgi complex in the context of surrounding organelles: ER, yellow; membrane-bound ribosomes, blue; free ribosomes, orange; MTs, bright green; dense core vesicles, bright blue; clathrin-negative vesicles, white; clathrin-positive compartments and vesicles, bright red; clathrin-negative compartments and vesicles, purple; mitochondria, dark green. Reprinted with permission from the authors and the Proceedings of the National Academy of Sciences. Bars, 500 nm.
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Figure 5: Stereo views of a model generated by tomographic reconstruction of a slab of cytoplasm from a cultured cell, strain HIT-T15. Three serial 400-nm sections were reconstructed by dual axis tomography, and the IMOD software package was used to model all visible objects within a volume 3.1 × 3.2 × 1.2 μm3 (see Marsh et al. 2001 for details). The model displays the Golgi complex in the context of surrounding organelles: ER, yellow; membrane-bound ribosomes, blue; free ribosomes, orange; MTs, bright green; dense core vesicles, bright blue; clathrin-negative vesicles, white; clathrin-positive compartments and vesicles, bright red; clathrin-negative compartments and vesicles, purple; mitochondria, dark green. Reprinted with permission from the authors and the Proceedings of the National Academy of Sciences. Bars, 500 nm.

Mentions: An additional advantage of EM tomography is that many cellular structures are visualized at once with a single imaging technology. This is a significant difference from fluorescence light microscopy, where one sees only that which has been made fluorescent. Indeed, it is this feature of immunofluorescence that allows one to be satisfied with fixation protocols that preserve some cellular details very badly (Melan and Sluder 1992). EM tomography of well-fixed cells, on the other hand, reveals the relationships among diverse cellular structures at comparatively high resolution (Fig. 5). The stereo images presented here show a 1.2-μm slab of cytoplasm from a cultured mammalian cell. The cisternae of the Golgi region and the ER are displayed with all the nearby vesicles, microtubules, and ribosomes (Marsh et al. 2001). This view reveals just how crowded cytoplasm really is. However, note that even though the model in Fig. 5 looks densely packed with organelles, fully 65% of the volume modeled lies outside of organelles, allowing plenty of space for cytosol.


Electron microscopy of cells: a new beginning for a new century.

McIntosh JR - J. Cell Biol. (2001)

Stereo views of a model generated by tomographic reconstruction of a slab of cytoplasm from a cultured cell, strain HIT-T15. Three serial 400-nm sections were reconstructed by dual axis tomography, and the IMOD software package was used to model all visible objects within a volume 3.1 × 3.2 × 1.2 μm3 (see Marsh et al. 2001 for details). The model displays the Golgi complex in the context of surrounding organelles: ER, yellow; membrane-bound ribosomes, blue; free ribosomes, orange; MTs, bright green; dense core vesicles, bright blue; clathrin-negative vesicles, white; clathrin-positive compartments and vesicles, bright red; clathrin-negative compartments and vesicles, purple; mitochondria, dark green. Reprinted with permission from the authors and the Proceedings of the National Academy of Sciences. Bars, 500 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192021&req=5

Figure 5: Stereo views of a model generated by tomographic reconstruction of a slab of cytoplasm from a cultured cell, strain HIT-T15. Three serial 400-nm sections were reconstructed by dual axis tomography, and the IMOD software package was used to model all visible objects within a volume 3.1 × 3.2 × 1.2 μm3 (see Marsh et al. 2001 for details). The model displays the Golgi complex in the context of surrounding organelles: ER, yellow; membrane-bound ribosomes, blue; free ribosomes, orange; MTs, bright green; dense core vesicles, bright blue; clathrin-negative vesicles, white; clathrin-positive compartments and vesicles, bright red; clathrin-negative compartments and vesicles, purple; mitochondria, dark green. Reprinted with permission from the authors and the Proceedings of the National Academy of Sciences. Bars, 500 nm.
Mentions: An additional advantage of EM tomography is that many cellular structures are visualized at once with a single imaging technology. This is a significant difference from fluorescence light microscopy, where one sees only that which has been made fluorescent. Indeed, it is this feature of immunofluorescence that allows one to be satisfied with fixation protocols that preserve some cellular details very badly (Melan and Sluder 1992). EM tomography of well-fixed cells, on the other hand, reveals the relationships among diverse cellular structures at comparatively high resolution (Fig. 5). The stereo images presented here show a 1.2-μm slab of cytoplasm from a cultured mammalian cell. The cisternae of the Golgi region and the ER are displayed with all the nearby vesicles, microtubules, and ribosomes (Marsh et al. 2001). This view reveals just how crowded cytoplasm really is. However, note that even though the model in Fig. 5 looks densely packed with organelles, fully 65% of the volume modeled lies outside of organelles, allowing plenty of space for cytosol.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA. richard.mcintosh@colorado.edu

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Thus, in spite of its value for antigen localization, the approach is not an ideal way to study cell morphology... Its reliance on both chemical fixation and infiltration with an osmotically active cryoprotectant reduces the likelihood that the resulting images will reflect the condition in a living cell... Finally, there are indications that an electron energy loss filter, developed by several companies to remove electrons that were inelastically scattered by the specimen before an image is formed, can enhance the quality of images from thick, frozen-hydrated specimens (Grimm et al. 1997)... Although these technologies have led to significant progress in imaging frozen cells, there are still formidable problems to overcome before this approach will yield the most useful results... These problems have recently been addressed by combining the power of RFFSE with tomography to obtain 3-D views of cellular structures that should closely resemble the living state... The accuracy of these specimen preparation methods has been tested in three ways: (a) significant fractions (1/20–1/5) of rapidly frozen yeast cells are viable upon rewarming, showing that rapid freezing itself preserves the native structure of cells very well. (b) RFFSE samples have been compared with equivalent ones prepared by freeze-fracture EM, a method that visualizes replicas of fractured cellular surfaces formed by metal shadowing at very low temperatures... These studies have found no significant difference between the structures seen by the two methods (e.g., Linder and Staehelin 1979). (c) The images from samples prepared by RFFSE conform to expectations that are based on a broad range of knowledge about subcellular components: cytoskeletal fibers usually run straight; membrane profiles of the ER and of Golgi cisternae appear turgid and smooth; most vesicles are round; cytomatrix is even; even chromatin appears structured (McDonald and Morphew 1993; Ladinsky et al. 1999; Muller et al. 2000)... Thus, studies that have melded RFFSE with tomography are likely to be showing us cellular structures that are essentially native, revealing organelle morphology in its normal context and in a situation where the parameters of cell physiology, like cell cycle stage, can be manipulated by the experimenter... An additional advantage of EM tomography is that many cellular structures are visualized at once with a single imaging technology... EM tomography of well-fixed cells, on the other hand, reveals the relationships among diverse cellular structures at comparatively high resolution (Fig. 5)... The tomograms of RFFSE samples already show enough detail and quality to prompt serious study in their own right, and it is realistic to think about using these methods to view major cellular subsystems, like an entire nucleus or a Golgi complex... Of more concern is the issue of recognizing macromolecules of interest within cellular tomograms... We see a brave new world emerging from the combination of modern EM with the rapidly advancing methods for light microscopy... The resulting data should help us to understand the context for functional genomics and elucidate the continuum of structural order that bridges from inanimate molecules to the order of the living cell.

Show MeSH
Related in: MedlinePlus