Electron microscopy fixation

Hyatt M A 1981 Changes in specimen volume Fixation for Electron Microscopy ed M A Hyatt (New York Academic) pp 299-306... 

Peachey L D, Ishikawa H and Murakami T 1996 Correlated confocal and intermediate voltage electron microscopy imaging of the same cells using sequential fluorescence labeling fixation and critical point dehydration Scanning Microsc. (SuppI) 10 237-47... 

Fig. 6. Electron microscopy of Ca -ATPase crystals in thin sections. Sarcoplasmic reticulum (2mg of protein/ml) was solubilized in the standard crystallization medium with C12E8 (2mg/mg protein) and incubated under nitrogen at 2°C for 15 days. The crystalline sediment was embedded in Epon-Araldite mixture and processed for electron microscopy. Depending on conditions during fixation, embedding, sectioning and viewing, the observed periodicities in different specimens varied between 103 and 147 A. Magnification, x 207000. From Taylor et al. [156].

III. Transmission electron microscopy of radish seeds Transmission electron microscopy (TEM) of radish seeds was done as listed below For TEM preparations, the specimens after fixation and dehydration, were embedded in Epon 812 resin (Luft, 1961). Thick sections (ca. 1mm each) were stained with 0.1% toluidine blue and observed with a Zeiss light photomicroscope. Thin sections, obtained with a diamond knife on a Supernova microtome, were sequentially stained at room temperature with 2% uranyle acetate (aqueous) for 5 min and by lead citrate for 10 min (Reynolds, 1963). Ultrastructural studies were made using a Philips CM12 transmission electrone microscope (TEM) operated at 80 KV. 

Chemical fixation for transmission electron microscopy prepares cells for the preservation of damage due to subsequent washing with aqueous solvents, dehydration with organic solvents such as ethanol or acetone, embedding in plastic resins, polymerization of the resins by heat, exothermic catalysts, or ultraviolet radiation, and imaging with high-energy electron beams in an electron microscope. 

Somljo It all depends on the fixation method. We use osmium ferricyanide to selectively infiltrate the SR. If we use intermediate high-voltage electron microscopy, we can look at thicker specimens, and this technique provides more extensive views than obtained from the usual thin sections. This is the same information we get when we infiltrate the SR with Fluo-3. These pictures are pretty reliable. Furthermore, if you want to confirm without chemical fixation, methods such as rapid freezing are available. All these techniques give the same pictures, which vary according to the smooth muscle type. 

Sanders With traditional electron microscopy the space looks as if it might be as short as 20 nm. Is this the same sort of volume that might be predicted by freeze fixation ... 

List of Fixation Conditions for Preparation of Plant Cells and Tissues for Transmission Electron Microscopy... 

As mentioned, chemical fixation of plant cells has been reviewed many times (15-20) and the reader is referred to these citations for a variety of fixation procedures for preserving plant cells and tissues. One of the most recent references regarding the topic is that of Hopwood and Milne (21). Table 1 presents their recommendations regarding fixation of plant cells and tissues for electron microscopy. 

Hopwood D, Milne G. Fixation, in Electron Microscopy in Biology. A Practical Approach (Harris JR, ed.), IRL Press, Oxford, UK, 1991, pp. 1-15. 

Powell, M. D., Speare, D. J. and Burka, J. F. (1992). Fixation of mucus on rainbow trout (Oncorhynchus mykiss Walbaum) gills for light and electron microscopy, J. Fish. Biol., 41, 813-824. 

Preparation of biological material for electron microscopy still required fixation, dehydration, and ultrathin sections. Araldite and other resins were used in place of paraffin wax for blocking. At first, specially sharpened steel knives were employed to cut the sections, but from 1950 glass or diamond knives were used which could cut slices 100-200 nm thick. By 1952, Palade and others were obtaining sections... 

Hyat, M. A. (1981) Fixation for Electron Microscopy. Academic, New York. 

Glauert, A. M. (1974) Fixation, dehydration and embedding of biological specimens, in Practical Methods in Electron Microscopy, vol. 3 (Glauert, A. M., ed.), North-Holland, Amsterdam, pp. 1-201. 

Leonard, J. B. and Shepardson, S. P. (1994) A comparison of heating modes in rapid fixation techniques for electron microscopy. J. Histochem. Cytochem. 42,383-391. 

Fixation in glutaraldehyde prodnces better morphology, but induces a great deal of autofluorescence, and limits cytoplasmic and nnclear permeability. A protocol utilizing glutaraldehyde followed by borohydride treatment has been previously described that is applicable also for electron microscopy of cultnred cells (4). Some areas of the cell are relatively impermeable with this approach, bnt this is an excellent choice of fixative protocol for microtnbnle morphology. 

Immunolabel samples either before or after fixation. Any size gold can be used for scanning electron microscopy. The size of the gold particles is limited by the resolution of the instrument. 

Login, G. R. and Dvorak, A. M. (1988) Microwave fixation provides excellent preservation of tissue, cells and antigens for light and electron microscopy. Histochem. J. 20, 373-387. 

Palade, Porter, and Sjostrand perfected thin sectioning and fixation methods for electron microscopy of intracellular structures. 

Curiously there have been few examples in the literature (outside of work with cereals) which use light microscopy of embedded sections as anything other than an adjunct to TEM or SEM [27,28]. Thus, fixation and embedding schedules relate to the electron microscopies ... 

FIGURE 1.1. Electron micrographs of liver tissue processed differently for transmission electron microscopy. (A) Processed by the conventional method. Note superior quality of ultrastructural preservation compared with that obtained with microwave heating. (B) Rapidly processed by vacuum microwave heating. The whole process from tissue fixation to resin embedding was completed in 2 hr. The quality of ultrastructural preservation is satisfactory. Magnification 6,21 Ox (B). (B) courtesy of Richard T. Giberson. 

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