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.

An Environmental Wet Cell For High Voltage Electron Microscopy

1973 ◽  
Vol 31 ◽  
pp. 18-19
Author(s):  
N.J. Tighe ◽  
H.M. Flower ◽  
P.R. Swann
Keyword(s):  
Electron Microscopy ◽  
High Temperature ◽  
Image Quality ◽  
Inelastic Scattering ◽  
High Voltage ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Environmental Cell ◽  
Water Saturated ◽  
High Temperature Gas

A differentially pumped environmental cell has been developed for use in the AEI EM7 million volt microscope. In the initial version the column of gas traversed by the beam was 5.5mm. This permited inclusion of a tilting hot stage in the cell for investigating high temperature gas-specimen reactions. In order to examine specimens in the wet state it was found that a pressure of approximately 400 torr of water saturated helium was needed around the specimen to prevent dehydration. Inelastic scattering by the water resulted in a sharp loss of image quality. Therefore a modified cell with an ‘airgap’ of only 1.5mm has been constructed. The shorter electron path through the gas permits examination of specimens at the necessary pressure of moist helium; the specimen can still be tilted about the side entry rod axis by ±7°C to obtain stereopairs.

The Network Parameters Of The T-System In Frog Muscle Measured With The High Voltage Electron Microscope.

1975 ◽  
Vol 33 ◽  
pp. 550-551 ◽  
Author(s):  
Brenda R. Eisenberg ◽  
Lee D. Peachey
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
High Voltage ◽  
Sartorius Muscle ◽  
High Voltage Electron Microscopy ◽  
Network Parameters ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
High Voltage Electron ◽  
T System

Analysis of the electrical properties of the t-system requires knowledge of the geometry of the t-system network. It is now possible to determine the network parameters experimentally by use of high voltage electron microscopy. The t-system was marked with exogenous peroxidase. Conventional methods of electron microscopy were used to fix and embed the sartorius muscle from four frogs. Transverse slices 0.5-1.0 μm thick were viewed at an accelerating voltage of 1000 kV using the JEM-1000 high voltage electron microscope at Boulder, Colorado and prints at x5000 were used for analysis.The length of a t-branch (t) from node to node (Fig. 1a) was measured with a magnifier; at least 150 t-branches around 30 myofibrils were measured from each frog. The mean length of t is 0.90 ± 0.11 μm and the number of branches per myofibril is 5.4 ± 0.2 (mean ± SD, n = 4 frogs).

Histo-grade diamond knives may be an appropriate tool for high-voltage electron microscopy

1991 ◽  
Vol 49 ◽  
pp. 296-297
Author(s):  
M.E. Rock ◽  
J.A. Anderson ◽  
P.S. Binder
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Stereo Pair ◽  
Specimen Thickness ◽  
Serial Sections ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Diamond Knife

High voltage electron microscopy (HVEM) has been employed in various ways (whole mounts of cells stereo pair imaging, axial tomography, and serial sections for reconstruction) to elucidate three dimensional (3-D) ultrastructural data. The increased specimen thickness allows further data analysis unobtainable from ultra-thin sections. HVEM can reduce the number of sections needed in 3-D reconstructiortby approximately ten times over conventional transmission electron microscopy (CTEM). But increasing section thickness also increases wear on the diamond knife used to section. We have compared the serial sections obtained from a histo-grade diamond knife with those from an E.M. grade ultra-knife. Both sets of sections were cut 0.5 μm thick from the same block, and evaluated under the one million volt beam of the HVEM.

The application of three-dimensional tomographic reconstruction methods to high-voltage electron microscopy

1987 ◽  
Vol 45 ◽  
pp. 570-573
Author(s):  
B. F. McEwen ◽  
C. L. Rieder ◽  
M. Radermacher ◽  
R. A. Grassucci ◽  
J. N. Turner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Three Dimensional ◽  
Depth Of Field ◽  
Specimen Thickness ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Reconstruction Methods

High-voltage electron microscopy (HVEM) has considerably increased the thickness limit of biological specimens that can be visualized at high resolution. Because of its increased penetration power, HVEM is potentially the most powerful tool available for obtaining three-dimensional (3D) information concerning the structure of cells. In the past, such information was primarily obtained from serial thin sections or techniques based on surface shadowing, but these methods have severe problems and limitations which can only be overcome by imaging greater depths in the samples (see refs. 1 and 2). HVEM has yet to realize its potential for 3D structural determination because of the confusion arising from the overlap of features at different depths in the sample. Due to the relatively large depth of field, which exceeds the specimen thickness, HVEM (like all electron microscopy) produces an image that is essentially a projection of the sample.

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.

Aperture Dependence of Resolution and Beam Transmission in High-Voltage Tem of Thick Biological Specimens

1976 ◽  
Vol 34 ◽  
pp. 564-565
Author(s):  
Mircea Fotino
Keyword(s):  
Electron Microscopy ◽  
Electron Energy ◽  
High Voltage ◽  
Cross Sections ◽  
Dominant Factor ◽  
Specimen Thickness ◽  
Transmitted Beam ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Half Angle

In standard transmission electron microscopy the image is formed by electrons scattered both elastically and inelastically within the specimen. Their relative contributions to the image quality are determined by electron energy, specimen thickness and vertex half angle a of the collection cone defined by the objective aperture.The cross sections for scattering into a given angle decrease as the electron energy increases to the MeV region (1), and the accompanying increased preponderance of the elastic component results in improved resolution in the image of thin objects.In most biological applications of high-voltage electron microscopy the usual thickness range (0.25-5.0 μn) is such that plural scattering within the specimen becomes the dominant factor for resolution and contrast (e.g.(2)). In particular, increased fractions of electrons are removed from the transmitted beam by scattering outside the collection cone.

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.

High Voltage Electron Microscopy in Biology

1970 ◽  
Vol 28 ◽  
pp. 12-13
Author(s):  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
Heavy Metal ◽  
Electron Microscope ◽  
High Voltage ◽  
Cell Structure ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Sectioning Method ◽  
High Voltage Electron ◽  
Electron Microscopes

Conventional electron microscopes operate with accelerating voltages up to 100kV. Because of the scattering of electrons by atoms of the specimen an image with reasonable resolution can only be obtained with very thin specimens. The study of cell structure with the electron microscope became possible only with the introduction of ultramicrotomes which produce sections of plastic-embedded tissues down to about 100A in thickness. It has long been known that useful images could be obtained with much thicker materials at higher accelerating voltages (cf. refs. 1 and 2) and in the early sixties electron microscopes operating at voltages of up to one million Volts were built in Japan and in France. Their capabilities were soon demonstrated in metallurgy but they were ignored until recently by biologists. For one, biologists were busy exploiting the sectioning method and in addition may have been deterred by the knowledge that scattering contrast rapidly decreases at higher accelerating voltage. Only recently has it been realized that excellent contrast is obtained at 500 and 1000kV with the usual heavy metal stains (3,4). High voltage microscopes are now manufactured commercially in England (AEI), France (GESPA) and Japan (Hitachi, Jeol) and should soon be more widely accessible.

Integrated microscopy resource for biomedical research at the university of wisconsin at madison

1987 ◽  
Vol 45 ◽  
pp. 594-597 ◽  
Author(s):  
G. Schatten ◽  
J. Pawley ◽  
H. Ris
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Transmission Electron Microscope ◽  
High Voltage ◽  
Biomedical Research ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
University Of Wisconsin ◽  
Transmission Electron ◽  
The University

The High Voltage Electron Microscopy Laboratory [HVEM] at the University of Wisconsin-Madison, a National Institutes of Health Biomedical Research Technology Resource, has recently been renamed the Integrated Microscopy Resource for Biomedical Research [IMR]. This change is designed to highlight both our increasing abilities to provide sophisticated microscopes for biomedical investigators, and the expansion of our mission beyond furnishing access to a million-volt transmission electron microscope. This abstract will describe the current status of the IMR, some preliminary results, our upcoming plans, and the current procedures for applying for microscope time.The IMR has five principal facilities: 1.High Voltage Electron Microscopy2.Computer-Based Motion Analysis3.Low Voltage High-Resolution Scanning Electron Microscopy4.Tandem Scanning Reflected Light Microscopy5.Computer-Enhanced Video MicroscopyThe IMR houses an AEI-EM7 one million-volt transmission electron microscope.

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