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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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Electron tomographic reconstruction of frozen-hydrated specimens. A and B are two 12-nm slices extracted from a reconstruction of the archaebacterium, Pyrodictium abyssi (Baumeister et al. 1999). The distance between the slices is 90 nm. The boundary of the cell is evident, including both the plasma membrane and the “S-layer,” which is composed of ordered protein subunits. There are also crystalline structures in the cytoplasm (square arrays in A and B). Vesicles within the cell are evident. The dark circle in each image is a 250-nm latex sphere, which is apparently being internalized by endocytosis. Reprinted with permission from the author and Trends in Cell Biology. C displays a slice from a tomographic reconstruction of isolated, frozen-hydrated thermosomes, which are chaperonins with eightfold rotational symmetry. The proteins are arranged in a top view orientation. Image processing methods have been used to search for sites of correlations between the tomogram shown in C and a template generated from high resolution cryo-EM micrographs of the thermosome. Positions of strong correlation are marked by white crosses in D. This method for feature recognition holds promise as a way to identify the location of specific proteins in tomographic images of cytoplasm. Reprinted with permission of the authors and the Proceedings of the National Academy of Sciences. Bar, (B) 200 nm.
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Figure 2: Electron tomographic reconstruction of frozen-hydrated specimens. A and B are two 12-nm slices extracted from a reconstruction of the archaebacterium, Pyrodictium abyssi (Baumeister et al. 1999). The distance between the slices is 90 nm. The boundary of the cell is evident, including both the plasma membrane and the “S-layer,” which is composed of ordered protein subunits. There are also crystalline structures in the cytoplasm (square arrays in A and B). Vesicles within the cell are evident. The dark circle in each image is a 250-nm latex sphere, which is apparently being internalized by endocytosis. Reprinted with permission from the author and Trends in Cell Biology. C displays a slice from a tomographic reconstruction of isolated, frozen-hydrated thermosomes, which are chaperonins with eightfold rotational symmetry. The proteins are arranged in a top view orientation. Image processing methods have been used to search for sites of correlations between the tomogram shown in C and a template generated from high resolution cryo-EM micrographs of the thermosome. Positions of strong correlation are marked by white crosses in D. This method for feature recognition holds promise as a way to identify the location of specific proteins in tomographic images of cytoplasm. Reprinted with permission of the authors and the Proceedings of the National Academy of Sciences. Bar, (B) 200 nm.

Mentions: The problem of superposed detail in frozen hydrated samples has been tackled by using multiple tilted views and the mathematics of tomography to build 3-D images of small cells and isolated organelles (Grimm et al. 1998; for review see Baumeister et al. 1999; Nicastro et al. 2000). Fig. 2A and Fig. B show slices extracted from a tomographic reconstruction of the archaebacterium, Pyrodictium abyssi. Here, automation of data collection has been used to keep the electron dose-per-view to a minimum, and the principle of dose-fractionation (McEwen et al. 1995) has allowed multiple tilted views to be assembled into a single 3-D volume whose signal-to-noise ratio is defined approximately by the total dose for the series, rather than the dose/image (subject to the constraint that each image must show enough detail to permit its accurate alignment with other tilted views). This work shows tremendous promise, given both the nearly native state of the cells at the time they are imaged and the 3-D detail that is visible. Objects separated by only 6–8 nm are visible in the best of these cryotomograms. At this resolution some large protein complexes can be recognized by their structure alone, suggesting that cryotomography followed by appropriate image analysis may provide information about the localization of specific proteins (Fig. 2C and Fig. D). However, note that the promising results in the latter two figures have so far been achieved with frozen protein solutions, not with cytoplasm itself (Boehm et al. 2000).


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

McIntosh JR - J. Cell Biol. (2001)

Electron tomographic reconstruction of frozen-hydrated specimens. A and B are two 12-nm slices extracted from a reconstruction of the archaebacterium, Pyrodictium abyssi (Baumeister et al. 1999). The distance between the slices is 90 nm. The boundary of the cell is evident, including both the plasma membrane and the “S-layer,” which is composed of ordered protein subunits. There are also crystalline structures in the cytoplasm (square arrays in A and B). Vesicles within the cell are evident. The dark circle in each image is a 250-nm latex sphere, which is apparently being internalized by endocytosis. Reprinted with permission from the author and Trends in Cell Biology. C displays a slice from a tomographic reconstruction of isolated, frozen-hydrated thermosomes, which are chaperonins with eightfold rotational symmetry. The proteins are arranged in a top view orientation. Image processing methods have been used to search for sites of correlations between the tomogram shown in C and a template generated from high resolution cryo-EM micrographs of the thermosome. Positions of strong correlation are marked by white crosses in D. This method for feature recognition holds promise as a way to identify the location of specific proteins in tomographic images of cytoplasm. Reprinted with permission of the authors and the Proceedings of the National Academy of Sciences. Bar, (B) 200 nm.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: Electron tomographic reconstruction of frozen-hydrated specimens. A and B are two 12-nm slices extracted from a reconstruction of the archaebacterium, Pyrodictium abyssi (Baumeister et al. 1999). The distance between the slices is 90 nm. The boundary of the cell is evident, including both the plasma membrane and the “S-layer,” which is composed of ordered protein subunits. There are also crystalline structures in the cytoplasm (square arrays in A and B). Vesicles within the cell are evident. The dark circle in each image is a 250-nm latex sphere, which is apparently being internalized by endocytosis. Reprinted with permission from the author and Trends in Cell Biology. C displays a slice from a tomographic reconstruction of isolated, frozen-hydrated thermosomes, which are chaperonins with eightfold rotational symmetry. The proteins are arranged in a top view orientation. Image processing methods have been used to search for sites of correlations between the tomogram shown in C and a template generated from high resolution cryo-EM micrographs of the thermosome. Positions of strong correlation are marked by white crosses in D. This method for feature recognition holds promise as a way to identify the location of specific proteins in tomographic images of cytoplasm. Reprinted with permission of the authors and the Proceedings of the National Academy of Sciences. Bar, (B) 200 nm.
Mentions: The problem of superposed detail in frozen hydrated samples has been tackled by using multiple tilted views and the mathematics of tomography to build 3-D images of small cells and isolated organelles (Grimm et al. 1998; for review see Baumeister et al. 1999; Nicastro et al. 2000). Fig. 2A and Fig. B show slices extracted from a tomographic reconstruction of the archaebacterium, Pyrodictium abyssi. Here, automation of data collection has been used to keep the electron dose-per-view to a minimum, and the principle of dose-fractionation (McEwen et al. 1995) has allowed multiple tilted views to be assembled into a single 3-D volume whose signal-to-noise ratio is defined approximately by the total dose for the series, rather than the dose/image (subject to the constraint that each image must show enough detail to permit its accurate alignment with other tilted views). This work shows tremendous promise, given both the nearly native state of the cells at the time they are imaged and the 3-D detail that is visible. Objects separated by only 6–8 nm are visible in the best of these cryotomograms. At this resolution some large protein complexes can be recognized by their structure alone, suggesting that cryotomography followed by appropriate image analysis may provide information about the localization of specific proteins (Fig. 2C and Fig. D). However, note that the promising results in the latter two figures have so far been achieved with frozen protein solutions, not with cytoplasm itself (Boehm et al. 2000).

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