Electron spectroscopic imaging of chemical versus cryo-fixed myocardial cells

1991 ◽  
Vol 49 ◽  
pp. 288-289
Author(s):  
Robyn Rufner ◽  
Rebecca C. Stearns ◽  
Cindy L. Hastings ◽  
James D. Marsh ◽  
John J. Godleski
Keyword(s):  
Freeze Drying ◽  
Osmium Tetroxide ◽  
Spectroscopic Imaging ◽  
Chemical Fixation ◽  
Myocardial Cells ◽  
Electron Spectroscopic Imaging ◽  
Freeze Dried ◽  
Processing Regimes ◽  
Electron Energy Loss ◽  
Intracellular Ions

Since chemical fixation and dehydration of cells results in loss and/or redistribution of endogenous ions, cryofixation with a process that preserves ultrastructure and the localization of intracellular ions is critical. Myocytes and macrophages prepared by the LifeCell® process of slam-freezing and freeze drying with subsequent osmium tetroxide/paraformaldehyde fixation have exhibited excellent structural and compositional integrity when examined by Electron Spectroscopic Imaging (ESI) and by Electron Energy Loss Spectroscopy (EELS). However, it has not been established if the apparent enhanced structure, particularly of cristae and membranes within mitochrondria, is solely a result of lipid preservation or if fixation with osmium tetroxide vapors can explain the pronounced “thickness” of membranes. In this study the effects of freezeslammed, freeze-dried myocytes with two different processing regimes for postfixation were compared to myocytes prepared by conventional chemical fixation.

Electron spectroscopic imaging of isolated myocardial cells prepared by slam-freeze molecular distillation processing

1990 ◽  
Vol 48 (3) ◽  
pp. 774-775
Author(s):  
Robyn Rufner ◽  
Rebecca C. Stearns ◽  
Cindy L. Hastings ◽  
Steven Borzak ◽  
John J. Godleski
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Molecular Distillation ◽  
Spectroscopic Imaging ◽  
Myocardial Cells ◽  
Electron Spectroscopic Imaging ◽  
Sprague Dawley ◽  
Freeze Dried

Isolated adult rat ventricular myocytes serve as a well-defined model to study subcellular events associated with cardiac ischemia. Optimal ultrastructural preservation of in vivo and cultured myocardial cells has been approached by cryo-preservation techniques involving rapid freezing and subsequent freeze substitution or freeze-dried cryosections. Alterations in biochemical structure and composition inherent with chemical fixation procedures can be alleviated by molecular distillation drying of cryo-fixed specimens. In this morphological study, the effects of metal-mirror cryofixation and molecular distillation processing of myocardial cells was evaluated using electron spectroscopic imaging.Myocardial cells were isolated from the ventricles of adult female Sprague-Dawley rats by enzymatic digestion. Briefly, perfused ventricles were minced and incubated with calcium-free Krebs-Henseleit buffer containing 0.05% collagenase and 0.03% hyaluroxidase with 0.002% deoxyribonuclease II and 0.002% trypsin and 1 mM Ca for 10-20 min at 37° with continuous oxygenation and shaking. The tissue was transferred to KH-enzyme buffer plus 2% bovine serum albumin, filtered through Nitex mesh, sedimented by centrifugation (150xg) and then resuspended in 1 mM Ca KH buffer with 2% bovine serum albumin. The yield of viable cells was greater than 70%.

Electron spectroscopic imaging and diffraction: applications II materials science

1992 ◽  
Vol 50 (2) ◽  
pp. 1198-1199
Author(s):  
J. Mayer
Keyword(s):  
Electron Microscopy ◽  
Energy Loss ◽  
Materials Science ◽  
Spectroscopic Imaging ◽  
Electron Spectroscopic Imaging ◽  
Imaging Spectrometer ◽  
Transmission Electron ◽  
Diffraction Patterns ◽  
Electron Spectroscopic Diffraction ◽  
Electron Energy Loss

With imaging energy filters becoming commercially available in transmission electron microscopy many of the limitations of conventional TEM instruments can be overcome. Energy filtered images of diffraction patterns can now be recorded without scanning using efficient parallel (2-dimensional detection. We have evaluated a prototype of the Zeiss EM 912 Omega, the first commercially available electron microscope with integrated imaging Omega energy filter. Combining the capabilities of the imaging spectrometer with the principal operation modes of a TEM gives access to many new qualitative and quantitative techniques in electron microscopy. The basis for all of them is that the filter selecte electrons within a certain energy loss range ΔE1 <ΔE < ΔE2 and images their contribution to an image (electron spectroscopic imaging, ESI) or a diffraction pattern (electron spectroscopic diffraction, ESD) In many applications the filter is only used to remove the inelastically scattered electrons (elastic or zero loss filtering). Furthermore, the electron energy loss spectrum can be magnified and recorded with serial or parallel detection.

Freeze-Drying-Embedding Method of Specimens For Analysis of Diffusible Elements by Electron Spectroscopic Imaging

1985 ◽  
Vol 43 ◽  
pp. 702-703
Author(s):  
G.T. Simon ◽  
E. Spitzer
Keyword(s):  
Freeze Drying ◽  
Spectroscopic Imaging ◽  
Analytical Techniques ◽  
Fixation Method ◽  
Primary Concern ◽  
Electron Spectroscopic Imaging ◽  
Critical Importance ◽  
Electron Microscopic ◽  
Quick Freezing ◽  
Entrance Velocity

Electron spectroscopic imaging (ESI), developed in the last decade by F. P. Ottensmeyer and his group is one of the most sensitive electron microscopic analytical techniques available today. Spatial resolutions of 0.5.m and mass resolution of 50 atoms or less can be achieved. The limitations of ESI resides in the fact that only very thin specimens (20nm in thickness) can be used. It is therefore impossible to analyze frozen-dried or frozen hydrated sections.The primary concern in analyzing diffusible elements is their extraction and translocation during processing. Already existing freeze-drying embedding techniques have been modified. Quick freezing is by far the fixation method of choice. The type of coolant and the entrance velocity are of critical importance. Vitrification layers of 25-30 microns can be obtained by plunging small fragments of tissue into liquid propane at an entrance velocity of more than 6.4 m/s.

Embedding of unfixed plant material in water-soluble resin: A simple preparation method for elemental analysis by ESI and EELS techniques

1990 ◽  
Vol 48 (3) ◽  
pp. 772-773
Author(s):  
Heiner Lehmann ◽  
Ulrike Kunz
Keyword(s):  
Preparation Method ◽  
Plant Cells ◽  
Inorganic Ions ◽  
Living Cells ◽  
Water Soluble ◽  
Preparation Technique ◽  
Chemical Fixation ◽  
Electron Spectroscopic Imaging ◽  
Simple Preparation ◽  
Electron Energy Loss

The localization of elements in cells and tissues by electron spectroscopic imaging (ESI) or electron energy loss spectroscopy (EELS) can be realized only in very thin specimens in order to avoid multiple scattering of the electrons. Therefore the biological material has to be cut in thin 30 nm sections.The commonly used preparation procedure is a hydrous chemical fixation followed by dehydration, embedding in hydrophobic resin and ultrathin sectioning. The disadvantage of this procedure is the loss and/or dislocation of inorganic ions and other water-soluble substances. To overcome these difficulties the preferable preparation method is cryofixation in combination with cryosubstitution, but this is a very expensive and time-wasting procedure.Meanwhile, we established a more simplified preparation technique, which shows reasonable results on plant cells without large central vacuoles: The living cells were directly put into freshly mixed Nanoplast FB 101, a water-soluble melanine resin, and stored for 24 h at 20° C in a silca gel containing exsiccator.

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