Cytoskeletal features of epithelia prepared by “extractive fixation“

1987 ◽  
Vol 45 ◽  
pp. 902-903
Author(s):  
J. M. Holy ◽  
J. A. Oaks
Keyword(s):  
Tannic Acid ◽  
Cultured Cells ◽  
Freeze Substitution ◽  
Fixation Method ◽  
High Voltage Electron Microscopy ◽  
Ionic Detergents ◽  
Voltage Electron ◽  
Ionic Detergent ◽  
Whole Mount ◽  
Background Material

Techniques such as whole mount (high-voltage) electron microscopy of cultured cells (frequently extracted with non-ionic detergents), rapid-freezing (followed either by freeze-substitution or deep etching and rotary-replication), and reversible embedment of tissue have furnished novel insights into cytoskeletal architecture. Another approach in the examination of cytoskeletal morphology has been the modification of routine fixation and thin-section methods. Minimizing osmium exposure and including tannic acid in the primary fixative has been found to improve cytoskeletal preservation. Maupin and Pollard developed a fixation method in which a non-ionic detergent is added to the fixative to facilitate penetration of tannic acid into the cytoplasm. The images obtained by tannic acid fixation are frequently very electron-dense, however, and it is often difficult to clearly visualize the cytoplasmic filamentous system. This study describes results of an attempt to extract background material and reduce the density resulting from tannic acid fixation by reducing the concentration of aldehydes in the primary fixative.

Whole Mount Observation of Cultured Cells

1980 ◽  
Vol 38 ◽  
pp. 802-805 ◽  
Author(s):  
K. Hama
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Image Quality ◽  
High Voltage ◽  
Cultured Cells ◽  
Specimen Thickness ◽  
High Voltage Electron Microscopy ◽  
Cellular Architecture ◽  
Voltage Electron ◽  
Whole Mount

The cellular architecture of cultured cells has been investigated on critical-point dried whole mount preparations with the aid of stereo-high voltage electron microscopy2,4,5. In these preparations, the absence of an embedding material permits an stereoobservation at rather low accelerating voltage1,3. In the present study, whole mount preparations of cultured chick fibroblasts were examined in the electron microscope operated at 100 KV, 200 KV, 500 KV, 750 KV and 1,000 KV to investigate the voltage dependency of the usable specimen thickness and of the image quality at different specimen thickness.

Use of Thick Histochemical Preparations, Whole Mount Cultured Cells or Thick Sections from Embedded Tissues, for Three-Dimensional Observation by Ultrahigh Voltage Electron Microscopy

1990 ◽  
Vol 48 (3) ◽  
pp. 184-185
Author(s):  
Tetsuji Nagata ◽  
Nobuteru Usuda ◽  
Hongjun Ma
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Rat Hepatocytes ◽  
Wistar Rat ◽  
Specific Cell ◽  
Cell Organelles ◽  
Voltage Electron ◽  
Culture Cells ◽  
Whole Mount

We have observed very thick biological specimens, both whole mount cultured cells and thick sections from embedded tissues, which were stained with histochemical reactions for specific cell organelles, by means of high or ultrahigh voltage electron microscopy (UHVEM).Thick histochemical specimens, both whole mount cultured cells and semithin sections from embedded tissues were used. Two cell strains in culture, one established CHO-K1 cell line and the other primary culture cells of adult Wistar rat hepatocytes were used. Culture cells were seeded onto formval coated gold meshes and incubated in CO2 incubator. CHO-K1 cells were cultured in Ham’s F12 medium containing HRP (1 mg/ml), while rat hepatocytes were in L-15 medium containing Clofibrate (0.2 mM). The cultured cells were stained with DAB reaction, dried in a critical point dryer (Hitachi HCP-1). Thick sections (0.2-1.0 μm) from DEHP fed rat livers, fixed in 2.5% glutaraldehyde, stained with DAB reaction, postfixed in 1% osmium tetroxide and embedded in Epon, or semithin Epon sections (0.2μm) from the pancreases of 3H-thymidine or 3H-uridine injected mice, fixed doubly, radioautographed with Sakura NR-H2 emulsion.

scholarly journals THREE-DIMENSIONAL OBSERVATIONS ON THICK BIOLOGICAL SPECIMENS BY HIGH VOLTAGE ELECTRON MICROSCOPY

2011 ◽  
Vol 19 (1) ◽  
pp. 51 ◽  
Author(s):  
Tetsuji Nagata
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Cell Organelles ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Thiamine Pyrophosphatase ◽  
Pinocytotic Vesicles

Thick biological specimens prepared as whole mount cultured cells or thick sections from embedded tissues were stained with histochemical reactions, such as thiamine pyrophosphatase, glucose-6-phosphatase, cytochrome oxidase, acid phosphatase, DAB reactions and radioautography, to observe 3-D ultrastructures of cell organelles producing stereo-pairs by high voltage electron microscopy at accerelating voltages of 400-1000 kV. The organelles demonstrated were Golgi apparatus, endoplasmic reticulum, mitochondria, lysosomes, peroxisomes, pinocytotic vesicles and incorporations of radioactive compounds. As the results, those cell organelles were observed 3- dimensionally and the relative relationships between these organelles were demonstrated.

Light microscopy of living cells correlative to high-voltage EM and low-voltage SEM of cell cryo-whole-mounts

1992 ◽  
Vol 50 (1) ◽  
pp. 566-567
Author(s):  
Marek Malecki
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
High Voltage ◽  
Living Cell ◽  
Low Voltage ◽  
Neoplastic Cell ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Cytoskeleton Reorganization ◽  
Whole Mount

Analysis of motility phenomena in a living cell observed with light microscopy can be significantly enriched by preparing a whole-mount of this cell for high voltage electron microscopy (to reveal the intracellular organization) and for low voltage scanning electron microscopy (to reveal the surface topography). In earlier studies, cell whole-mount prepration by chemical fixation and drying was adequate for studies of slow cellular motions at the subcellular level (e.g. receptor movements). Fast cellular motions analysed at the supramolecular level (e.g. transmitter release, cytoskeleton reorganization) required development of much faster cryo-immobilization methods. However, in studies of cells grown on grids, these freezing methods involved time consuming transfer of these cells , from an incubator to a freezer, making impossible fine correlations between images of a living cell and its cryo-whole-mount. To overcome this constraint for correlative microscopical studies of neoplastic cell motility, I designed an instrument consisting of a freezer attached to a light microscope and allowing cryoimmobilization within miliseconds after recording. The main objective of the current project was refinement of an instrument and improvement of appropriate specimen cryo-preparation techniques.

Studies on The Cytoplasmic Ground Substance Using High Voltage Electron Microscopy of Whole Cultured Cells

1980 ◽  
Vol 38 ◽  
pp. 818-821
Author(s):  
K.R. Porter ◽  
K.J. Luby
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Monolayer Culture ◽  
Cultured Cells ◽  
Ground Substance ◽  
High Quality ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Cytoplasmic Ground Substance ◽  
Very High

Cells of several types, when grown and maintained in monolayer culture, will spread on the substrate to be not greater than 1 pm thick in their thinner margins. When fixed with glutaraldehyde and OsO4 and then dried by the critical-point method,these cells can be viewed in the HVEM and stereo images of very high quality can be obtained. Grown directly on formvar-coated gold grids, such cells are quickly and easily prepared for microscopy.

scholarly journals The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

1985 ◽  
Vol 100 (5) ◽  
pp. 1474-1487 ◽  
Author(s):  
H Ris
Keyword(s):  
Critical Point ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Intact Cells ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Cytoplasmic Filaments ◽  
Critical Point Drying ◽  
Cytoplasmic Filament ◽  
Whole Mounts

High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.

Resources for the Study of Cellular Structure by High Voltage Electron Tomography, Serial Thin Sectioning, Specific Labeling, and Image Analysis

1997 ◽  
Vol 3 (S2) ◽  
pp. 273-274
Author(s):  
David Mastronarde ◽  
James Kremer ◽  
Eileen O’Toole ◽  
Mary Morphew ◽  
Mark Ladinsky ◽  
...  
Keyword(s):  
High Voltage ◽  
Cultured Cells ◽  
Electron Tomography ◽  
Freeze Substitution ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
High Quality ◽  
Isotropic Resolution ◽  
Voltage Electron ◽  
Tissue Specimens

We are working to improve methods for the study of cellular fine structure. Our approach is to advance each of the key steps in the preparation of specimens for EM: high quality fixation that will preserve both structure and antigenicity; methods for specific labeling; efficient acquisition of 3-D electron microscopic data; and software for 3-D reconstruction and display.Our work on high quality structure preservation has focused on methods for fast freezing and freeze substitution. Both plunge freezing of specimens grown on coated gold grids and high pressure freezing of either cultured cells or tissue specimens have yielded well preserved material. These samples are suitable for freeze substitution fixation with either anhydrous aldehydes in acetone at -90°C, for the preservation of antigens, or aldehydes, tannic acid, OsO4, and uranyl acetate for optimal preservation the structure.We have used a JEOL JEM-1,000 high voltage microscope to image sections about 250nm thick, employing a goniometer stage to perform dual axis tomography for 3-D reconstruction with approximately isotropic resolution at ∼7nm.

scholarly journals Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells.

1984 ◽  
Vol 98 (6) ◽  
pp. 2126-2132 ◽  
Author(s):  
M C Beckerle
Keyword(s):  
Tissue Culture ◽  
Cultured Cells ◽  
Cytoplasmic Matrix ◽  
High Voltage Electron Microscopy ◽  
Polystyrene Beads ◽  
Voltage Electron ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
A Cell ◽  
Path Lengths

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.

High Voltage Electron Microscopy of Ion-Milled Dental Enamel

1974 ◽  
Vol 32 ◽  
pp. 60-61
Author(s):  
L. D. Ackerman ◽  
S. H. Y. Wei
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Third Molar ◽  
Polarized Light ◽  
Dental Enamel ◽  
Longitudinal Section ◽  
Ion Milling ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron

Mature human dental enamel has presented investigators with several difficulties in ultramicrotomy of specimens for electron microscopy due to its high degree of mineralization. This study explores the possibility of combining ion-milling and high voltage electron microscopy as a means of circumventing the problems of ultramicrotomy.A longitudinal section of an extracted human third molar was ground to a thickness of about 30 um and polarized light micrographs were taken. The specimen was attached to a single hole grid and thinned by argon-ion bombardment at 15° incidence while rotating at 15 rpm. The beam current in each of two guns was 50 μA with an accelerating voltage of 4 kV. A 20 nm carbon coating was evaporated onto the specimen to prevent an electron charge from building up during electron microscopy.

A High Voltage Electron Microscopy Study of Sub-Oxide Formation in Vanadium

1971 ◽  
Vol 29 ◽  
pp. 212-213
Author(s):  
R. H. Geiss ◽  
R. L. Ladd ◽  
K. R. Lawless
Keyword(s):  
High Vacuum ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Ultra High Vacuum ◽  
Pure Oxygen ◽  
Pressure Oxidation ◽  
High Voltage Electron Microscopy ◽  
Oxidation Study ◽  
Voltage Electron ◽  
Good Agreement

Detailed electron microscope and diffraction studies of the sub-oxides of vanadium have been reported by Cambini and co-workers, and an oxidation study, possibly complicated by carbon and/or nitrogen, has been published by Edington and Smallman. The results reported by these different authors are not in good agreement. For this study, high purity polycrystalline vanadium samples were electrochemically thinned in a dual jet polisher using a solution of 20% H2SO4, 80% CH3OH, and then oxidized in an ion-pumped ultra-high vacuum reactor system using spectroscopically pure oxygen. Samples were oxidized at 350°C and 100μ oxygen pressure for periods of 30,60,90 and 160 minutes. Since our primary interest is in the mechanism of the low pressure oxidation process, the oxidized samples were cooled rapidly and not homogenized. The specimens were then examined in the HVEM at voltages up to 500 kV, the higher voltages being necessary to examine thick sections for which the oxidation behavior was more characteristic of the bulk.

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