Sandwich freezing device for rapid freezing of viruses, bacteria, yeast, cultured cells and animal and human tissues in electron microscopy

Microscopy ◽  
2020 ◽  
Author(s):  
Masashi Yamaguchi ◽  
Masaki Taguchi ◽  
Katsuyuki Uematsu ◽  
Azusa Takahashi-Nakaguchi ◽  
Michiyo Sato-Okamoto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Freeze Substitution ◽  
Biological Materials ◽  
Rapid Freezing ◽  
Human Tissues ◽  
New Device ◽  
Wide Range ◽  
Excellent Preservation

Abstract We have been using sandwich freezing of living yeast and bacteria followed by freeze-substitution for observing close-to-native ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells and human tissues have been found to give excellent preservation of ultrastructure of cells and tissues. These studies, however, have been conducted using a handmade sandwich freezing device and have been limited in a few laboratories. To spread the use of this method to other laboratories, we fabricated and commercialized a new sandwich freezing device. The new device is inexpensive, portable and sterilizable. It can be used to rapid-freeze viruses, bacteria, yeast, cultured cells and animal and human tissues to a depth of 0.2 mm if tissues are prefixed with glutaraldehyde. The commercial availability of this device will expand application of rapid freezing to wide range of biological materials.

Preparation of dipteran larvae for scanning electron microscopy using a freeze-substitution technique

1986 ◽  
Vol 64 (3) ◽  
pp. 797-799 ◽  
Author(s):  
Douglas D. Colwell ◽  
Eric G. Kokko
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Fixation Methods ◽  
Good Preservation ◽  
Larval Stages ◽  
Excellent Preservation ◽  
Standard Fixation ◽  
Scanning Electron

Difficulties are often encountered in achieving good preservation of dipteran larvae for scanning electron microscopy. Standard fixation methods frequently fail because fixatives do not penetrate the cuticle rapidly, resulting in distortion and generation of artifacts on the surface. A freeze-substitution technique that produces excellent preservation of the larval stages of a wide variety of Diptera is described. The method employs Freon 12 for rapid freezing of the specimens and methanol as the substitution fluid. The technique is simple, inexpensive, and demonstrates a significant improvement in preservation of specimens.

scholarly journals PB-10 High-pressure freezing method can be replaced by sandwich freezing method !?: electron microscopy of human tissues and cultured cells

Microscopy ◽  
2019 ◽  
Vol 68 (Supplement_1) ◽  
pp. i50-i50
Author(s):  
Masashi Yamaguchi ◽  
Seiichiro Wakabayashi ◽  
Yuumi Nakamura ◽  
Hiroyuki Matsue ◽  
Takuya Hirao ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Cultured Cells ◽  
Human Tissues ◽  
High Pressure Freezing ◽  
Freezing Method

Bacterial envelope profiles revealed by freeze-substitution

1992 ◽  
Vol 50 (1) ◽  
pp. 868-869
Author(s):  
L.L. Graham ◽  
T.J. Beveridge
Keyword(s):  
Electron Microscopy ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Chemical Fixation ◽  
Diverse Range ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Conventional Methods ◽  
Section Electron Microscopy ◽  
Better Than

Traditional methods of processing bacteria for thin section electron microscopy rely on chemical fixation and dehydration under conditions which maximize specimen deterioration. Cryotechniques, however, use physical fixation (rapid freezing) and are slowly being recognized as a superior alternative to the more conventional methods. Freeze-substitution is a cryotechnique which combines cryofixation with a gentle chemical fixation and dehydration regimen, yielding specimens amenable to standard embedment procedures and ultramicrotomy. Previous study has shown that freeze-substitution retains the molecular composition of eubacteria better than conventional methods of processing. In this study we extend our observations and show that a simple freeze-substitution protocol reliably preserves the ultrastructure of a diverse range of microorganisms including archaeobacteria and anaerobic eubacteria.

scholarly journals Rapid Freezing/Freeze Substitution Transmission Electron Microscopy Assessment of Cultured Endothelial Cell Glycocalyx Structure

2011 ◽  
Vol 17 (S2) ◽  
pp. 162-163
Author(s):  
E Ebong ◽  
F Macaluso ◽  
D Spray ◽  
J Tarbell
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endothelial Cell ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Transmission Electron

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

scholarly journals Good Ultrastructural Preservation of Human Tissues and Cultured Cells by Glutaraldehyde Fixation, Sandwich Freezing, and Freeze-Substitution

CYTOLOGIA ◽  
2020 ◽  
Vol 85 (1) ◽  
pp. 15-26
Author(s):  
Masashi Yamaguchi ◽  
Seiichiro Wakabayashi ◽  
Yuumi Nakamura ◽  
Hiroyuki Matsue ◽  
Takuya Hirao ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Freeze Substitution ◽  
Human Tissues ◽  
Glutaraldehyde Fixation

scholarly journals Evidence for "twisted plane" undulations in golden hamster sperm tails

1977 ◽  
Vol 75 (3) ◽  
pp. 851-865 ◽  
Author(s):  
DM Woolley
Keyword(s):  
Electron Microscopy ◽  
Low Temperature ◽  
Ethylene Glycol ◽  
Golden Hamster ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
The Third ◽  
Working Hypothesis ◽  
Angular Displacements

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.

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