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.

Three Dimensional Immuno-Localization of Green Fluorescent Protein Chimeras Using Rapid Freezing and Freeze-Substitution

2000 ◽  
Vol 6 (S2) ◽  
pp. 298-299
Author(s):  
Mary Morphew ◽  
David Mastronarde ◽  
Eileen O'Toole ◽  
Mark Ladinsky ◽  
Brad Marsh ◽  
...  
Keyword(s):  
Low Temperature ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Cell Types ◽  
Uranyl Acetate ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Green Fluorescent ◽  
Structural Preservation

All microscopy is limited by the quality of the specimen under study. Three-dimensional (3-D) visualization of antigen localization using the electron microscope (EM) is particularly challenging due to the need to maintain the activity of some epitopes while preserving cellular ultrastructure. We have used rapid freezing to immobilize all cellular constituents almost instantaneously. Freeze-substitution of the frozen samples was used to stabilize the specimen and to accomplish low-temperature dehydration, minimizing perturbation of cellular structure. We have found that high pressure freezing, double jet freezing and plunge freezing are all useful for achieving high quality structural preservation for some cell types or for particular applications. For immunolocalization, we have had most success freeze-substituting into acetone containing 0.2% glutaraldehyde and 0.1 % uranyl acetate. We have utilized low-temperature acrylic embedding resins, Lowicryl HM20 and LRGold, to further maintain structure and decrease protein insolubility. Both of these resins have proven suitable for cutting serial thin sections.

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.

Low-temperature Scanning Electron Microscope constructed with high-excitation objective lens

1989 ◽  
Vol 47 ◽  
pp. 734-735
Author(s):  
H. Koike ◽  
T. Inoué
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Low Temperature ◽  
Scanning Electron Microscope ◽  
Freeze Substitution ◽  
Objective Lens ◽  
Rapid Freezing ◽  
High Excitation ◽  
Temperature Scanning ◽  
Scanning Electron

Low temperature microscope technology can be traced back to the last century including the time of light microscope, and its history is over a hundred years. In the field of electron microscopy, low temperature techniques such as the freeze-fracture replica, freeze-sectioning, freeze-substitution, etc. were tested up to early 1960s. According to the progress of the rapid-freezing method, the freeze-substitution and freeze-etching replica methods have provided great successful results.The low temperature scanning electron microscope (LTSEM) was also tested by Echlin et al. in 1970, and thereafter, a number of LTSEM constructions were attempted. These LTSEMs are generally classified into two groups: the type with fracturing and coating facilities directly attached to the SEM column, and the group having a separated preparation chamber and a transfer device. The LTSEM so far constructed were reviewed comprehensively in greater detail. Some such instruments were designed taking account of stringent requirements of low temperature techniques. These systems, however, seemed to be too comprehensive, involving complex procedures as compared withe their resolutions. In comparison with the conspicuous results obtained by other low temperature techniques, the LTSEM can be regarded as still in the stage prior to practical application from the viewpoint of the high resolution. In consideration of these circumstances, the present paper aims at providing a new LTSEM to realize simple operation retaining the advantage of the ultrastructural preservation by the rapid-freezing and the high resolution by introducing the high-excitation objective lens.

scholarly journals Immunoelectron microscopy of acetylcholine receptors and 43 KD protein after rapid freezing, freeze-substitution, and low-temperature embedding in lowicryl K11M.

1991 ◽  
Vol 39 (5) ◽  
pp. 625-634 ◽  
Author(s):  
G W Phillips ◽  
P C Bridgman
Keyword(s):  
Monoclonal Antibodies ◽  
Low Temperature ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Uv Light ◽  
Freeze Substitution ◽  
Rapid Freezing ◽  
Cytoplasmic Surface ◽  
Cytoplasmic Proteins ◽  
Anti Acetylcholine

To label intracellular determinants of the acetylcholine receptor and associated cytoplasmic proteins while preserving optimal ultrastructure, we developed a post-embedment labeling technique that uses rapid-frozen specimens and freeze-substitution without chemical fixatives. This procedure has been made possible through the use of a low-temperature resin (Lowicryl K11M) that can be polymerized with UV light at -60 degrees C. Rapid-frozen muscle cells were used to evaluate the preservation of structure, and Torpedo electroplaque cells and purified postsynaptic membranes were used to quantitatively evaluate the labeling specificity, efficiency, and resolution of the technique. The labeling efficiency of seven different monoclonal antibodies (MAb) to the acetylcholine receptor varied from 3-13%; there was a correlation between the degree of efficiency and the number of epitopes with which the antibodies reacted. The resolution of the technique was not sufficient to determine whether the anti-acetylcholine receptor MAb were bound to the cytoplasmic or the extracellular surface, but was sufficient to correctly determine the location of the receptor-associated 43 KD protein on the cytoplasmic surface.

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.

Freeze substitution studies on cultured human fibroblasts

1989 ◽  
Vol 47 ◽  
pp. 1008-1009
Author(s):  
Julian P. Heath ◽  
Donna Turner
Keyword(s):  
Osmium Tetroxide ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Three Dimensional ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Lung Fibroblasts ◽  
Rapid Freezing ◽  
Thin Sections ◽  
Cultured Human Fibroblasts

We are using rapid freezing and freeze substitution to study the three dimensional organisation of membrane systems and cytoskeletal filaments in motile fibroblasts. This study has two objectives: first, to provide material for structural and immunocytochemical analysis of membrane-cytoskeletal interactions in cells that have been preserved with minimum artefact (1,2,3) and second, to refine and develop existing rapid freezing and freeze substitution techniques to allow for the study of single cells that have been experimentally manipulated and observed by digital video microscopy before fixation.The cells used were human lung fibroblasts (IMR90) either growing on Lux Thermanox coverslips or as pelleted suspensions. The cells were slam frozen on a Med-Vac Cryo Press against a liquid nitrogen cooled copper block. Coverslips were trimmed to 2 x 2 mm in size, excess fluid was drained off, and they were placed on top of a 1mm thick gelatin cushion on an aluminium planchette. For cell suspensions, 3 ul was placed on top of the gelatin cushion. Frozen samples were placed in acetone containing 1% osmium tetroxide for 72 hours at 192 K, wanned to 253 K for 4 hours, and then brought to room temperature. The samples were rinsed in acetone and embedded in Spurr’s resin. Thin sections were cut on a RMC6000 ultramicrotome, stained in uranyl acetate and Reynolds' lead citrate and photographed on a Philips EM410 electron microscope at 60 keV.

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