High‐pressure freezing for the preservation of biological structure: Theory and practice

Dahl, Rolf; Staehelin, L. Andrew

doi:10.1002/jemt.1060130305

Cited by 313 publications

(190 citation statements)

References 25 publications

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“…During cryofixation optimal structural preservation requires that the specimen be frozen so rapidly that cellular water forms vitreous (i.e., glass-like) rather than crystalline ice (Dahl and Staehelin 1989;Studer et al 1989). The latter causes an extensive and unmistakable disruption of structure that is worse than the effects of conventional fixation protocols.…”

Section: Resultsmentioning

confidence: 99%

“…Rapid vitreous freezing followed by FS is generally considered to be the method of choice for faithfully preserving cell structure at the highest possible resolution (reviewed in Moor 1987;Dahl and Staehelin 1989;Studer et al 1989;McDonald 1994McDonald , 1998. During the freezing process internal and external cellular constituents, including their soluble components, are immobilized in their native structure and position in just a few milliseconds.…”

Section: Discussionmentioning

confidence: 99%

“…A method based on rapid freezing followed by freeze substitution (FS) is now available that allows thicker specimens, including vertebrate cells in mitosis, to be processed for EM with minimal structural alterations (reviewed in Moor 1987;Dahl and Staehelin 1989;McDonald and Morphew 1993;Studer et al 1995;McDonald 1998). This procedure relies on using a high-pressure freezing (HPF) apparatus to prevent ice crystal formation during freezing (i.e., vitreous freezing).…”

Section: Introductionmentioning

confidence: 99%

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A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

et al. 1998

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Three decades of structural analysis have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque outer plate that is separated by a light-staining electrontranslucent middle plate from an inner plate associated with the surface of the pericentric heterochromatin. Spindle microtubules terminate in the outer plate and, in their absence, a conspicuous corona of fine filaments radiates from the cytoplasmic surface of this plate. Here we report for the first time the ultrastructure of kinetochores in untreated and Colcemid-treated vertebrate somatic (PtK 1 ) cells prepared for optimal structural preservation using high-pressure freezing and freeze substitution. In serial thin sections, and electron tomographic reconstructions, the kinetochore appears as a 50-75 nm thick mat of light-staining fibrous material that is directly connected with the more electron-opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and is surrounded on its cytoplasmic surface by a conspicuous 100-150 nm wide zone that excludes ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light-staining, thin (<9 nm diameter) fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms, which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since the middle plate is not visible in these kinetochore preparations this feature is likely an artifact produced by extraction and coagulation during conventional fixation and/or dehydration procedures.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

et al. 1998

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“…The specimen carriers were placed in the holder of a high-pressure freezer (HPM010, BAL-TEC) and shock frozen as described (Hohenberg et al, 1994). Freeze substitution with Osmium tetroxide, embedding in Epoxy resin, and thin sectioning with a cryo-ultramicrotom were performed as described elsewhere (Dahl and Staehelin, 1989;El-Kest and Marth, 1992;Hohenberg et al, 1994). Electron microscopy of the frozen-hydrated ultrathin sections was performed in a Zeiss EM 912 microscope at a temperature of 100 K (-173°C).…”

Section: Transmission Electron Microscopymentioning

confidence: 99%

Listeria monocytogenesl‐forms respond to cell wall deficiency by modifying gene expression and the mode of division

Dell'Era

Buchrieser

Couvé³

et al. 2009

Molecular Microbiology

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SummaryCell wall-deficient bacteria referred to as L-forms have lost the ability to maintain or build a rigid peptidoglycan envelope. We have generated stable, nonreverting L-form variants of the Gram-positive pathogen Listeria monocytogenes, and studied the cellular and molecular changes associated with this transition. Stable L-form cells can occur as small protoplast-like vesicles and as multinucleated, large bodies. They have lost the thick, multilayered murein sacculus and are surrounded by a cytoplasmic membrane only, although peptidoglycan precursors are still produced. While they lack murein-associated molecules including Internalin A, membraneanchored proteins such as Internalin B are retained. Surprisingly, L-forms were found to be able to divide and propagate indefinitely without a wall. Time-lapse microscopy of fluorescently labelled L-forms indicated a switch to a novel form of cell division, where genome-containing membrane vesicles are first formed within enlarged L-forms, and subsequently released by collapse of the mother cell. Array-based transcriptomics of parent and L-form cells revealed manifold differences in expression of genes associated with morphological and physiological functions. The L-forms feature downregulated metabolic functions correlating with the dramatic shift in surface to volume ratio, whereas upregulation of stress genes reflects the difficulties in adapting to this unusual, cell wall-deficient lifestyle.

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“…High pressure freezing (HPF) is a method for freezing isolated organelles, cells and tissues up to ~200-500 µm in depth without significant ice crystal damage (Dahl and Staehelin, 1989;McDonald, 1999;Moor, 1987;Shimoni and Muller, 1998). When cells are rapidly frozen, all contents are immobilized almost immediately.…”

Section: Introductionmentioning

confidence: 99%

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

Sosinsky

Crum

Jones

et al. 2008

Journal of Structural Biology

115

114

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The emergence of electron tomography as a tool for three dimensional structure determination of cells and tissues has brought its own challenges for the preparation of thick sections. High pressure freezing in combination with freeze substitution provides the best method for obtaining the largest volume of well-preserved tissue. However, for deeply embedded, heterogeneous, labile tissues needing careful dissection, such as brain, the damage due to anoxia and excision before cryofixation is significant. We previously demonstrated that chemical fixation prior to high pressure freezing preserves fragile tissues and produces superior tomographic reconstructions compared to equivalent tissue preserved by chemical fixation alone. Here, we provide further characterization of the technique, comparing the ultrastructure of Flock House Virus infected DL1 insect cells that were 1) high pressure frozen without fixation, 2) high pressure frozen following fixation, and 3) conventionally prepared with aldehyde fixatives. Aldehyde fixation prior to freezing produces ultrastructural preservation superior to that obtained through chemical fixation alone that is close to that obtained when cells are fast frozen without fixation. We demonstrate using a variety of nervous system tissues, including neurons that were injected with a fluorescent dye and then photooxidized, that this technique provides excellent preservation compared to chemical fixation alone and can be extended to selectively stained material where cryofixation is impractical.

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High‐pressure freezing for the preservation of biological structure: Theory and practice

Cited by 313 publications

References 25 publications

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

Listeria monocytogenesl‐forms respond to cell wall deficiency by modifying gene expression and the mode of division

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

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