scholarly journals HIGH RESOLUTION AUTORADIOGRAPHY WITH STRIPPING FILM

1971 ◽  
Vol 19 (5) ◽  
pp. 304-309 ◽  
Author(s):  
JOSEPH R. WILLIAMSON ◽  
HANK VAN DEN BOSCH
Keyword(s):  
High Resolution ◽  
Mechanical Device ◽  
Electron Microscopic ◽  
High Quality ◽  
Simple Method ◽  
Thin Sections ◽  
Large Numbers ◽  
High Resolution Autoradiography ◽  
Potential Doubling

A simple method is described for the preparation and use of stripping film of high quality for electron microscopic applications of autoradiography. The advantages of the method are: ( a) uniform monolayers of emulsion are easily prepared with an inexpensive mechanical device and applied to thin sections on conventional grids; ( b) both sides of grids can be coated with emulsion, permitting a potential doubling of sensitivity with, theoretically, no loss of resolution; and ( c) large numbers of grids can be processed simultaneously, assuring identical conditions of development.

scholarly journals HIGH-RESOLUTION AUTORADIOGRAPHY

1962 ◽  
Vol 15 (2) ◽  
pp. 173-188 ◽  
Author(s):  
Lucien G. Caro ◽  
Robert P. van Tubergen
Keyword(s):  
High Resolution ◽  
Silver Halide ◽  
Electron Microscopic ◽  
Simple Method ◽  
Electron Microscopic Autoradiography ◽  
Fine Grain ◽  
High Resolution Autoradiography ◽  
Silver Halide Crystals ◽  
Subcellular Level ◽  
Physical Developer

Methods used in obtaining high resolution in autoradiography, with special emphasis on the technique of electron microscopic autoradiography, are described, together with control experiments designed to establish the optimum conditions or procedures. On the basis of these experiments the emulsion selected was Ilford L-4, with a crystal size slightly larger than 0.1 micron. It is applied to the specimen in the form of a gelled film consisting of a monolayer of silver halide crystals. Background, when present, can be eradicated by a simple method. The preparations can be stored, in presence of a drying agent, at room temperature or in a refrigerator. Photographic development is done in Microdol, or in a special fine grain "physical" developer. For examination in the electron microscope the sections are stained with uranyl or lead stains. These methods give a good localization of the label, at the subcellular level, and good reproducibility in relative grain counts.

The High Resolution Appearance of Biological Substrates

1969 ◽  
Vol 27 ◽  
pp. 416-417
Author(s):  
Glen B. Haydon
Keyword(s):  
High Resolution ◽  
Phase Image ◽  
Biological Structure ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Resolution Electron ◽  
Discrete Structures ◽  
Large Phase ◽  
Biological Substrates ◽  
High Resolution Electron Micrographs

High resolution electron microscopic study of negatively stained macromolecules and thin sections of tissue embedded in a variety of media are difficult to interpret because of the superimposed phase image granularity. Although all of the information concerning the biological structure of interest may be present in a defocused electron micrograph, the high contrast of large phase image granules produced by the substrate makes it impossible to distinguish the phase ‘points’ from discrete structures of the same dimensions. Theory predicts the findings; however, it does not allow an appreciation of the actual appearance of the image under various conditions. Therefore, though perhaps trivial, training of the cheapest computer produced by mass labor has been undertaken in order to learn to appreciate the factors which affect the appearance of the background in high resolution electron micrographs.

Mise en évidence du rôle, dans la régénération des Amphibiens, d'une glycoprotéine sécrétée par la cape apicale: étude cytochimique et autoradiographique en microscopie électronique

Development ◽  
1974 ◽  
Vol 32 (1) ◽  
pp. 133-145
Author(s):  
Par Claude Chapron
Keyword(s):  
High Resolution ◽  
Epithelial Cells ◽  
Limb Regeneration ◽  
Target Cells ◽  
Silver Particles ◽  
Electron Microscopic ◽  
Microscopic Studies ◽  
High Resolution Autoradiography

Evidence for the role of an apical cap glycoprotein in amphibian regeneration: cytochemical and autoradiographic electron-microscopic studies Early during limb regeneration in the newt, an ectodermal apical cap covering a mesodermal blastema is formed. High-resolution autoradiography of these tissues has been carried out after incorporation of [3H]fucose, which is a precursor of glycoproteins. Autoradiography shows that silver particles are located at first on epithelial cells, then on mesenchymatous cells. This observation is consistent with a hypothesis in which the apical cap would elaborate a glycoprotein acting on the blastema. Substructural autoradiography and cytochemistry also show the importance of cellular surfaces for both cells producing glycoprotein and those which are target cells.

scholarly journals ALKALINE BISMUTH REAGENT FOR HIGH RESOLUTION ULTRASTRUCTURAL DEMONSTRATION OF PERIODATE-REACTIVE SITES

1972 ◽  
Vol 20 (12) ◽  
pp. 995-1005 ◽  
Author(s):  
STERLING K. AINSWORTH ◽  
MORRIS J. KARNOVSKY ◽  
SUSUMU ITO
Keyword(s):  
High Resolution ◽  
Periodic Acid ◽  
Periodate Oxidation ◽  
Ultrastructural Localization ◽  
Blocking Agent ◽  
Hydroxyl Groups ◽  
Electron Microscopic ◽  
Periodic Acid Schiff ◽  
Thin Sections ◽  
Bismuth Subnitrate

A simple technique is described for the ultrastructural localization of periodate-reactive mucosubstances and polysaccharides containing 1,2-glycols in thin sections of routinely fixed tissues. In this method the sugar residues are oxidized by periodic acid and the resulting aldehydes presumably reduce chelated bismuth subnitrate to metallic bismuth which then appears as a fine electron-opaque precipitate at the sites of the reducing sugars. The periodic acid-alkaline bismuth procedure provides a high resolution electron microscopic technique for demonstrating tissue sites of periodate-engendered groups very similar to the light microscopic periodic acid-Schiff reaction. The reaction can be prevented by the omission of periodate oxidation or alkaline bismuth subnitrate and by aldehyde blockage with the blocking agent, m-aminophenol. However, glycogen stains markedly without prior periodate oxidation, presumably through chelation of bismuth by hydroxyl groups. Other structures which stain without prior periodate oxidation are liver lysosomal dense bodies and, occasionally, ribosomes.

scholarly journals Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

1994 ◽  
Vol 126 (4) ◽  
pp. 901-910 ◽  
Author(s):  
T J Deerinck ◽  
M E Martone ◽  
V Lev-Ram ◽  
D P Green ◽  
R Y Tsien ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Substantial Improvement ◽  
Electron Microscopic ◽  
Simple Method ◽  
High Voltage Electron Microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

Three-dimensional reconstruction of large subcellular structures: A method for combining axial tilt tomography with serial reconstruction of thick sections

1992 ◽  
Vol 50 (1) ◽  
pp. 904-905
Author(s):  
Gabriel E. Soto ◽  
Maryann E. Martone ◽  
Stephan Lamont ◽  
Bridget O. Carragher ◽  
Thomas J. Deerinck ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Thin Sections ◽  
3 Dimensional ◽  
Voltage Electron ◽  
Large Numbers ◽  
Dimensional Reconstruction ◽  
Axial Tilt

The study of subcellular structures requires the resolution afforded by the electron microscope. However, cellular organelle systems can extend for tens of microns and therefore cannot be encompassed in a single thin section required for conventional electron microscopic observation. Even with the use of high voltage electron microscopy, section thickness is limited to no more than a few microns. Visualization of 3-dimensional cellular structure in large volumes of tissue can be achieved by using 3-dimensional reconstructions based on serial sections. This approach is often tedious, requiring an extremely large series of thin sections in order to encompass the structure of interest. This method also suffers from technical difficulties in obtaining, processing and maintaining adequate registration over large numbers of sections. We have been exploring a method in which the number of sections is reduced by employing a series of thick sections in which the structures of interest are selectively stained. Three-dimensional information is extracted from each section using axial tilt tomography. The resulting serial volumes are then aligned and linked to form a single volume which is displayed using volume rendering techniques.

scholarly journals LOSS OF PROTEINS AND OTHER MACROMOLECULES DURING PREPARATION OF CELL CULTURES FOR HIGH RESOLUTION AUTORADIOGRAPHY QUANTITATION BY A MICROMETHOD

1970 ◽  
Vol 18 (8) ◽  
pp. 565-573 ◽  
Author(s):  
TAPANI VANHA-PERTTULA ◽  
PHILIP M. GRIMLEY
Keyword(s):  
Amino Acids ◽  
High Resolution ◽  
Cell Cultures ◽  
Protein Extraction ◽  
Electron Microscopic ◽  
Human Carcinoma ◽  
Electron Microscopic Autoradiography ◽  
Binding Effect ◽  
Aldehyde Fixation ◽  
High Resolution Autoradiography

A reproducible procedure was devised in order to measure the extraction of 3H-labeled cellular products during aldehyde fixation and subsequent processing for electron microscopic autoradiography. Human carcinoma cell monolayers were cultivated in combustible plastic wells, so that all label could be counted as 3H2O. Radioisotope extraction during individual steps of processing could be analyzed and separate groups of experiments were directly comparable. Initial aldehyde fixation and subsequent buffer washes caused the major loss of radiolabeled amino acids, but this never exceeded 15% under conventional conditions. Radioisotope losses were influenced by the relative duration of fixation and buffer washes, fixation temperature and fixative concentration. Formaldehyde and glutaraldehyde both produced a nonspecific, time-related binding effect when 3H-labeled amino acids were introduced along with the fixative. Less significant nonspecific binding was observed when 3H-mannose, 3H-uridine or 3H-thymidine was added. Extraction of radioisotopes during formaldehyde fixation of cell cultures labeled with protein precursors was consistently greater than during glutaraldehyde fixation. Differences were less marked with the other precursors. Evaluation of the total protein extraction is complex, since the net losses observed were apparently the sum of precursor extraction, nonspecific amino acid binding and real molecular extraction. The implications for quantitative interpretative interpretation of high resolution autoradiography must be considered.

scholarly journals A Simple Method To Make High Resolution Projection Slides of Electron Micrographs

1994 ◽  
Vol 2 (6) ◽  
pp. 17-17 ◽  
Author(s):  
Jan S. Ryerse
Keyword(s):  
High Resolution ◽  
Electron Micrographs ◽  
Traditional Method ◽  
High Quality ◽  
Simple Method ◽  
Glass Slides ◽  
June July ◽  
The Cost

The traditional method of preparing high quality projection slides of one's best electron micrographs was to “print” negatives onto Kodak 2″ x 2″ emulsified glass slides using a standard enlarger fitted with a reduction lens and then to tray develop the slide to the desired contrast. As A. Kent Christensen pointed out in the June/July 1994 issue of this publication, the cost of 2″ x 2″ slides has become prohibitive. In any event, I always found adjusting the reduction lens on the enlarger to be a major hassle and searching for the correct exposure and developing times to be a ffustratingly inexact experience with much plate wastage.

scholarly journals A simple and reliable method for correlative light and electron microscopic studies.

1993 ◽  
Vol 41 (5) ◽  
pp. 769-772 ◽  
Author(s):  
J DeFelipe ◽  
A Fairén
Keyword(s):  
Golgi Method ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Simple Method ◽  
Thin Sections ◽  
Axonal Arborization ◽  
Accurate Localization ◽  
Mouse Cerebral Cortex ◽  
Microscopic Studies ◽  
Flat Embedding

We describe in detail a simple method for flat-embedding that can be subsequently used in correlative light and electron microscopic studies. The method can be applied to any material suitable for electron microscopy and is especially useful for study of the synaptology and ultrastructural characteristics of immunocytochemically or morphologically identified neurons or their processes. We present here an example to show how accurately one can delineate the fine details of a complex axonal arborization impregnated with the Golgi method in the mouse cerebral cortex. Golgi-impregnated sections to be studied at the electron microscopic level are osmicated, dehydrated, infiltrated with Araldite resin, flat-embedded, and identified cells or processes photographed. Serial semi-thin sections (1-2 microns thick) are then cut with an ultramicrotome, examined with the light microscope, and the elements rephotographed. Selected semi-thin sections are then resectioned on the ultramicrotome at 60-70 nm and examined electron microscopically. This method allows the systematic and accurate localization of stained cells and processes throughout the successive steps of the procedure.

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