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.

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.

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.

Three-Dimensional Visualization of the T-System of Frog Muscle Using High Voltage Electron Microscopy and a Lanthanum Stain

1977 ◽  
Vol 35 ◽  
pp. 570-571 ◽  
Author(s):  
Lee D. Peachey ◽  
Clara Franzini-Armstrong
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Three Dimensional ◽  
Biological Tissues ◽  
High Voltage Electron Microscopy ◽  
Transverse Tubular System ◽  
Peroxidase Reaction ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
T System

The effective study of biological tissues in thick slices of embedded material by high voltage electron microscopy (HVEM) requires highly selective staining of those structures to be visualized so that they are not hidden or obscured by other structures in the image. A tilt pair of micrographs with subsequent stereoscopic viewing can be an important aid in three-dimensional visualization of these images, once an appropriate stain has been found. The peroxidase reaction has been used for this purpose in visualizing the T-system (transverse tubular system) of frog skeletal muscle by HVEM (1). We have found infiltration with lanthanum hydroxide to be particularly useful for three-dimensional visualization of certain aspects of the structure of the T- system in skeletal muscles of the frog. Specifically, lanthanum more completely fills the lumen of the tubules and is denser than the peroxidase reaction product.

Radiation-Induced Precipitation at Thin Foil Surfaces in Ni-Be Alloys

1982 ◽  
Vol 40 ◽  
pp. 598-599
Author(s):  
T. Mukai ◽  
T. E. Mitchell
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Electron Irradiation ◽  
High Vacuum ◽  
Thin Foil ◽  
Homogeneous Precipitation ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Radiation Induced

Radiation-induced homogeneous precipitation in Ni-Be alloys was recently observed by high voltage electron microscopy. A coupling of interstitial flux with solute Be atoms is responsible for the precipitation. The present investigation further shows that precipitation is also induced at thin foil surfaces by electron irradiation under a high vacuum.

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.

High-voltage electron microscopy of maxillary gland of spirochete-infected brine shrimp: lesion of efferent tubule

1988 ◽  
Vol 46 ◽  
pp. 362-363
Author(s):  
G. E. Tyson ◽  
M. J. Song
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Brine Shrimp ◽  
Natural Populations ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
High Voltage Electron ◽  
Infected Adult

Natural populations of the brine shrimp, Artemia, may possess spirochete- infected animals in low numbers. The ultrastructure of Artemia's spirochete has been described by conventional transmission electron microscopy. In infected shrimp, spirochetal cells were abundant in the blood and also occurred intra- and extracellularly in the three organs examined, i.e. the maxillary gland (segmental excretory organ), the integument, and certain muscles The efferent-tubule region of the maxillary gland possessed a distinctive lesion comprised of a group of spirochetes, together with numerous small vesicles, situated in a cave-like indentation of the base of the tubule epithelium. in some instances the basal lamina at a lesion site was clearly discontinuous. High-voltage electron microscopy has now been used to study lesions of the efferent tubule, with the aim of understanding better their three-dimensional structure.Tissue from one maxillary gland of an infected, adult, female brine shrimp was used for HVEM study.

On the High Voltage Electron Microscopy (1 MeV) of Biological Thick Sections

1972 ◽  
Vol 30 ◽  
pp. 464-465
Author(s):  
William H. Massover
Keyword(s):  
Electron Microscopy ◽  
High Voltage ◽  
Present Report ◽  
Biological Tissues ◽  
Specimen Preparation ◽  
Technical Problems ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
Voltage Electron ◽  
High Voltage Electron

Stereoscopic examination of thick sections of fixed and embedded biological tissues by high voltage electron microscopy has been shown to allow direct visualization of three-dimensional fine structure. The present report will consider the occurrence of some new technical problems in specimen preparation and Image interpretation that are not common during lower voltage studies of thin sections.Thick Sectioning and Tissue Coloration - Epon sections of 0.5 μm or more that are cut with glass knives do not have a uniform thickness as Judged by their interference colors; these colors change with time during their flotation on the knife bath, and again when drying onto the specimen support. Quoted thicknesses thus must be considered only as rough estimates unless measured in specific regions by other methods. Chloroform vapors do not always result in good spreading of thick sections; however, they will spread spontaneously to large degrees after resting on the flotation bath for several minutes. Ribbons of thick sections have been almost impossible to obtain.

“High Resolution High Voltage Electron Microscopy”

1968 ◽  
Vol 26 ◽  
pp. 326-327
Author(s):  
Benjamin M. Siegel
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
High Voltage ◽  
Full Potential ◽  
Contrast Imaging ◽  
High Voltage Electron Microscopy ◽  
Low Contrast ◽  
Voltage Electron ◽  
Critical Areas ◽  
High Voltage Electron

The potential advantages of high voltage electron microscopy for extending the limits of resolution and contrast in imaging low contrast objects, such as biomolecular specimens, is very great. The results of computations will be presented showing that at accelerating voltages of 500-1000 kV it should be possible to achieve spacial resolutions of 1 to 1.5 Å and using phase contrast imaging achieve adequate image contrast to observe single atoms of low atomic number.The practical problems associated with the design and utilization of the high voltage instrument are, optimistically, within the range of competence of the state of the art. However, there are some extremely important and critical areas to be systematically investigated before we have achieved this competence. The basic electron optics of the column required is well understood, but before the full potential of an instrument capable of resolutions of better than 1.5 Å are realized some very careful development work will be required. Of great importance for the actual achievement of high resolution with a high voltage electron microscope is the fundamental limitation set by the characteristics of the high voltage electron beam that can be obtained from the accelerator column.

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