An Integrated Biological Imaging Facility: Capabilities of the Biological Microscopy and Image Reconstruction Resource

1997 ◽  
Vol 3 (S2) ◽  
pp. 271-272
Author(s):  
J. Frank ◽  
C.A. Mannella ◽  
C. Rieder
Keyword(s):  
Electron Microscopy ◽  
Image Reconstruction ◽  
3D Structure ◽  
Biological Imaging ◽  
Cell Manipulation ◽  
Projection Data ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Current Area

The Biological Microscopy and Image Reconstruction Resource (BMIRR) is operated by the Wadsworth Center as a national biotechnology resource, with funding through the NIH Center for Research Resources and from NSF. This biological imaging resource has evolved continuously over the past two decades. Early development focussed on correlative, same-cell light and electron microscopic techniques, combining the capabilities of video-enhanced light microscopy and high-voltage electron microscopy. A current area of development is electron microscopic tomography, whereby the full 3D capabilities of higher voltage (400-1200 KV) electron microscopy is brought to bear on biological problems. In particular, the recent development of techniques for merging projection data from two mutually perpendicular tilt series has permitted significantly improved resolution, reducing the missing wedge of information to a missing pyramid. Attention is now turning to optimization of conditions for applying tomography to frozen-hydrated specimens, using automated data collection on our cryo-IVEM. Combined with parallel advances in same-cell manipulation and viewing, the BMIRR provides biologists with a unique combination of imaging and computational tools for research into the 3D structure and dynamics that underly cellular processes (see figures and refs. 2-4).

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Correlated 3D Light and Electron Microscopy of Large, Complex Structures: Analysis of Transverse Tubules in Heart Failure

1998 ◽  
Vol 4 (S2) ◽  
pp. 440-441
Author(s):  
Maryann E. Martone ◽  
Andrea Thor ◽  
Stephen J. Young ◽  
Mark H. Ellisman.
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Light And Electron Microscopy ◽  
Structural Features ◽  
Electron Microscopic Level ◽  
Specimen Preparation ◽  
Large Sample Size ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron

Light microscopic imaging has experienced a renaissance in the past decade or so, as new techniques for high resolution 3D light microscopy have become readily available. Light microscopic (LM) analysis of cellular details is desirable in many cases because of the flexibility of staining protocols, the ease of specimen preparation and the relatively large sample size that can be obtained compared to electron microscopic (EM) analysis. Despite these advantages, many light microscopic investigations require additional analysis at the electron microscopic level to resolve fine structural features.High voltage electron microscopy allows the use of relatively thick sections compared to conventional EM and provides the basis for excellent new methods to bridge the gap between microanatomical details revealed by LM and EM methods. When combined with electron tomography, investigators can derive accurate 3D data from these thicker specimens. Through the use of correlated light and electron microscopy, 3D reconstructions of large cellular or subcellular structures can be obtained with the confocal microscope,

Three-dimensional imaging and physiology of live neurons and glia: Confocal light and correlative high-voltage Electron Microscopy

1993 ◽  
Vol 51 ◽  
pp. 162-163
Author(s):  
J.N. Turner ◽  
D.H. Szarowski ◽  
W. Shain ◽  
M. Davis-Cox ◽  
D.O. Carpenter ◽  
...  
Keyword(s):  
Large Scale ◽  
Culture Media ◽  
Shape Change ◽  
Complex Function ◽  
3D Structure ◽  
Three Dimensional ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
And Function

Correlating physiologic measures with three-dimensional (3D) imaging at the light and electron microscopic levels is a powerful combination of methods for studying the structure and function of biological systems. Neurobiology is an ideal field for the application of these methods because neurons and glia have complex and extensive 3D structure, and their physiology is under intense study. Neurons, such as those studied here from Aplysia, can be more than 100 μm in diameter, and glia undergo large scale 3D shape change as a function of a number of physiologic parameters. The ability to accurately quantitate the 3D structure, volume and surface area of live neurons and glia is important to our understanding of the complex function of these cells.Neurons were isolated from the major ganglia of juvenile Aplysia Californica and glia were obtained from long term cultures of LRM 55 cells or as primary isolates from rats. Cultures were exposed to Dil dissolved in DMSO with or without 20% Pluronic F-127 and added to the culture media. The imaging instrument was an Olympus IMT-2 and a Bio-Rad MRC-600.

Image Reconstruction Applied to High Voltage Microscopy

1974 ◽  
Vol 32 ◽  
pp. 396-397
Author(s):  
D. E. Johnson ◽  
J. Pfeifer
Keyword(s):  
Electron Microscopy ◽  
Image Reconstruction ◽  
High Voltage ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Three Dimensional Reconstruction ◽  
High Voltage Electron Microscopy ◽  
Voltage Electron ◽  
Dimensional Reconstruction ◽  
Quantitative Results

Greatly increased specimen penetration, which is the principle advantage of high voltage electron microscopy, carries with it an increased need for techniques to interpret the large amount of three-dimensional information projected into two-dimensional micrographs. Stereo views can provide very useful information and are widely used. However, for the general specimen, stereo views are limited in their ability to produce quantitative results. At the high voltage microscope facility, Univ. of Wisconsin, we have begun a program to develop and apply three dimensional reconstruction techniques to the microscopy of thick specimens.

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.

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