An excursion through the ultrastructural world of quick‐frozen pancreatic islets

Dudek, Ronald W.; Boyne, Alan F.

doi:10.1002/aja.1001750209

Cited by 34 publications

(25 citation statements)

References 60 publications

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“…Ultrarapid freezing provides a more accurate means of determining the subcellular distribution of antigens (Tokuyasu, 1986) including hormones (Dudek & Boyne, 1986) and a higher resolution of morphological details (Hirokawa & Kirino, 1980;Tatsuoka & Reese, 1989). Furthermore, fixatives such 03004864/91 $03.00 +.12 9 1991 Chapman and Hall Ltd.…”

Section: Introductionmentioning

confidence: 99%

Electron microscopic and immunocytochemical study of rapidly frozen, freeze-substituted neural lobes of rats

1991

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Rapid freezing of freshly dissected or incubated neural lobes was explored as a means of obtaining ultrastructural preservation of the more natural state of this tissue. A quantitative assessment of the region of good fixation was made in order to determine the relative fractions occupied by axons, pituicytes and the extracellular space. The immunocytochemical distributions of neurophysins and the glycopeptide portion of the vasopressin precursor were evaluated using the immunogold technique in order to determine the relative numbers of oxytocin and vasopressin fibre types in the fixed region, and the subcellular localization of these antigens. The uncut surface of rat neural lobes was rapidly frozen against a highly polished copper plug and freeze-substituted in osmium-acetone either immediately after dissection (approximately 2 min), or after a 15 min incubation period in vitro in an oxygenated, balanced salt solution. Substituted neural lobes were prepared for either conventional electron microscopy, or for immunogold labelling of neurophysins and the glycopeptide precursor to vasopressin. Membranes, subcellular organelles and extracellular matrix were well preserved 10 microns deep to the contacted surface. The extracellular space accounted for approximately 30% of the cross-sectional area of the neuropil and could be divided into two domains: an extended perivascular space (28-29% of total area); and a narrow (approximately 24 nm; approximately 1% of total) space between closely apposed neurosecretory processes or between these processes and pituicytes. Pituicytes accounted for about 30% of the area and axons 20-25%. Pituicytes occupied close to 60% of the basal lamina at the neurohaemal contact zone, while axons occupied approximately 20%. There were no differences between neural lobes frozen immediately after dissection and those incubated for 15 min in any of these measures, suggesting minimal fluid redistribution. Gold particles were specifically localized over large (100-200 nm) dense core vesicles, and less frequently over multivesicular bodies and lysosomes. No etching of the plastic or reduction of osmium was necessary to achieve labelling. Specific labelling of one set of terminals and axons (about 80%) was observed with the monoclonal antibody previously shown to be specific for oxytocin-neurophysin, while in neighbouring sections the remaining 20% of the processes were labelled with the antiserum to the vasopressin precursor, or with non-specific antibodies to neurophysins. In conclusion, ultrarapid freezing preserves a large extracellular space in the neural lobe and provides for high resolution morphological and immunocytochemical studies of neurohypophysial structure.

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Section: Introductionmentioning

confidence: 99%

Electron microscopic and immunocytochemical study of rapidly frozen, freeze-substituted neural lobes of rats

1991

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“…The superficial layer, where the impact takes place and which is best frozen (Onberg and Reese, 1979;Phillips and Boyne, 1984), is considerably compressed. This has been demonstrated for freeze-substituted specimens of brain slices N a n Harreveld and Fifkova, 1975), pancreatic islets (Dudek and Boyne, 1986), and olfactory epithelium . On the basis of freeze-substitution data, the mucus layer covering the olfactory epithelium surface in samples that are prepared with the slam-freeze method of has about half the thickness of that in samples not frozen with an impact method .…”

Section: Rapid-and High Pressure-freeze Results and Artifactsmentioning

confidence: 88%

“…Freeze-etching has been used for the finestructural investigation of extracellular environments, such as mucous fibers . For sections, freeze-substitution is superior to fixation at room temperature or around 5°C: (1) less material is washed out and the technique improves on (2) the outlines of structures, (3) the structural relationship between organelles, and the (4) retention of antigenicity (see also below under c; Dudek and Boyne, 1986;Phillips and Boyne, 1984;Sandoz et al, 1985;Steinbrecht, 1980). Freezesubstitution preserves lipid structures, e.g., honeycomb structures with local semi-fusion points, which cannot be discerned with other methods of thin-section electron microscopy.…”

Section: Cold Geniusmentioning

confidence: 98%

A survey of ultra‐rapid cryofixation methods with particular emphasis on applications to freeze‐fracturing, freeze‐etching, and freeze‐substitution

Menco

1986

J. Elec. Microsc. Tech.

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“…Secondly, in electron energy loss spectroscopy (EELS), electrons which have lost a specific amount of energy can be selected using an adjustable slit to build an image of elemental distributions within a sample. In the 1980s, EELS was frequently used by biologists for elemental mapping, with particular application to the composition of regulated secretory granules and elemental distribution along the regulated secretory pathway [27,56].…”

Section: Advances In Sample Preparation Techniquesmentioning

confidence: 99%

Towards native-state imaging in biological context in the electron microscope

2009

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Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context.

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An excursion through the ultrastructural world of quick‐frozen pancreatic islets

Cited by 34 publications

References 60 publications

Electron microscopic and immunocytochemical study of rapidly frozen, freeze-substituted neural lobes of rats

Electron microscopic and immunocytochemical study of rapidly frozen, freeze-substituted neural lobes of rats

A survey of ultra‐rapid cryofixation methods with particular emphasis on applications to freeze‐fracturing, freeze‐etching, and freeze‐substitution

Towards native-state imaging in biological context in the electron microscope

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