A preparation method for observing intracellular structures by scanning electron microscopy

Tánaka, Keiichi; Mitsushima, Akira

doi:10.1111/j.1365-2818.1984.tb00487.x

Cited by 175 publications

(123 citation statements)

References 12 publications

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“…The A-O-D-O method developed by TANAKA and MITSUSHIMA (1984) is a particularly effective method for such observations. By this method, fixed specimens are fractured into two pieces using the dimethyl sulf oxide (DMSO) freeze-cracking method originally developed by TOKUNAGA et al (1974), and usually only one side of the specimen is subjected to observations as per our routine works.…”

Section: Discussionmentioning

confidence: 99%

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Complementary Scanning Electron Microscopy: Technical Notes and Applications.

Inoué

1992

Archives of Histology and Cytology

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Summary.This report introduces practical techniques and applications of complementary scanning electron microscopy (SEM). To identify the complementary structures at high magnification, we first made a montage pair of low magnification micrographs as a guide map. Consulting the map, we took complementary micrographs at high magnification. When taking a picture at high magnification, we drew the outline of the most prominent structures on a transparent plastic plate attached to the cathode-ray tube of a SEM. Then another picture of the complementary structures was taken after adjusting the complementary structures to the reverse image of the plastic plate.We performed three applications of the complementary SEM: 1) complementary observation of the epithelial underside and lamina propria of the rat urinary bladder; 2) complementary observation of the fractured Golgi apparatus; and 3) evaluation of specimen drying methods.After proper digestion of the rat urinary bladder using strong alkali, the epithelium was detached from the underlying lamina propria. On the basal side of the epithelium, the meshwork of grooves were visible. The observation of the corresponding lamina propria confirmed that the grooves were occupied by blood capillaries located in the uppermost part of the lamina propria.The complementary observation of the Golgi apparatus was useful for understanding its three-dimensional architecture. The observation on both complementary fractured surfaces of the Golgi apparatus from the mouse lacrimal gland demonstrated the continuity of the Golgi stacks. It was also effective for observing both the cis and trans side of the same Golgi stack.Complementary SEM was also useful for evaluating the specimen drying method. One fractured specimen obtained by freeze-cracking was dried by the criticalpoint drying (CPD) method, and another complementary specimen was dried by the t-butyl freeze-drying method (BFD). The precise comparison of the preservation of the endoplasmic reticulum of pancreatic acinar cells indicated that the BFD technique preserved the ultrastructures better than CPD.

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Section: Discussionmentioning

confidence: 99%

“…The mouse lacrimal gland was used, and specimens were fundamentally prepared by the A-O-D-O method (TANAKA and MITSUSHIMA, 1984). After fixation with a mixture of 0.5% glutaraldehyde and 0.5% formaldehyde by perfusion, two split specimens were obtained by the DMSO freeze-cracking method.…”

Section: Complementary Observation Of the Fractured Golgi Apparatusmentioning

confidence: 99%

Complementary Scanning Electron Microscopy: Technical Notes and Applications.

Inoué

1992

Archives of Histology and Cytology

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“…Compact regions consist of piled stacks of Golgi cisterns with convex (cis-) and concave (trans-) sides that are linked to each other by a sparse tubular network defined as the non-compact region (Klumperman, 2011;Rambourg and Clermont 1990). Observations of osmium-macerated specimens under a high resolution scanning electron microscope has also revealed the diversity of the overall three-dimensional ultrastructure of the Golgi apparatus in various mammalian cells (Koga and Ushiki, 2006;Tanaka and Fukudome, 1991;Tanaka and Mitsushima, 1984;Tanaka et al, 1986). However, the profound mechanisms that promote structural diversity of the Golgi apparatus in vivo remain to be solved.…”

Section: Introductionmentioning

confidence: 99%

Functional implications of the Golgi and microtubular network in gonadotropes

Watanabe

Bochimoto

Koga

et al. 2014

Molecular and Cellular Endocrinology

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In contrast to the widely accepted images of the Golgi apparatus as a cup-like shape, the Golgi in pituitary gonadotropes is organized as a spherical shape in which the outer and inner faces are cis-and trans-Golgi elements, respectively. At the center of the spherical Golgi, a pair of centrioles is situated as a microtubule-organizing center from which radiating microtubules isotropically extend toward the cell periphery. This review focuses on the significance of the characteristic organization of the Golgi and microtubule network in gonadotropes, considering the roles of microtubule-dependent membrane transport in the formation and maintenance of the Golgi structure. Because the highly symmetrical organization of the Golgi is possibly perturbed in response to experimental treatments of gonadotropes, monitoring of the Golgi structure in gonadotropes under various experimental conditions will be a novel in vivo approach to elucidate the biogenesis of the Golgi apparatus.

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“…Chemical fixation of tissues was done by immersion in either buffered glutaraldehyde, or osmium tetroxide, or both. Other than immersion, tissues such as lung and kidney have been successfully fixed by perfusion fixation (Jongebloed andKalicharan 1994, Tanaka andMitsushima 1984). In other cases, single cells are embedded in fibrin gels (Haggis and Bond 1978) or in chitosan (Fukudome and Tanaka 1986) prior to fixation.…”

Section: Introductionmentioning

confidence: 99%

Field‐emission scanning electron microscopy of the internal cellular organization of fungi

et al. 2000

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Summary:Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular-and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.

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A preparation method for observing intracellular structures by scanning electron microscopy

Cited by 175 publications

References 12 publications

Complementary Scanning Electron Microscopy: Technical Notes and Applications.

Complementary Scanning Electron Microscopy: Technical Notes and Applications.

Functional implications of the Golgi and microtubular network in gonadotropes

Field‐emission scanning electron microscopy of the internal cellular organization of fungi

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