Fixation microscopy

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].

Fig. 2.1.6 Unassembled NMR microscopy probe with dedicated 15 mm resonator for expanded temperature ranges between — 100 and +200 °C (probe base, glass dewar, rf resonator, temperature sensor and fixation parts).

Body fluid specimens will be prepared and stained and the morphologic characteristic of the cells and the environment in which these cells are found will be examined by light microscopy. To achieve this, a representative cell sample must be obtained and adequate cell fixation is a prerequisite. Proper identification of the specimen and protection of the specimen s integrity are essential. Finally, pertinent patient clinical history is important for accurate specimen interpretation. 

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. 

Plant Cells and Tissues Structure-Function Relationships. Methods for the Cytochemical/Histochemical Localization of Plant Cell/Tissue Chemicals. Methods in Light Microscope Radioautography. Some Fluorescence Microscopical Methods for Use with Algal, Fungal, and Plant Cells. Fluorescence Microscopy of Aniline Blue Stained Pistils. A Short Introduction to Immunocytochemistry and a Protocol for Immunovi-sualization of Proteins with Alkaline Phosphatase. The Fixation of Chemical Forms on Nitrocellulose Membranes. Dark-Field Microscopy and Its Application to Pollen Tube Culture. Computer-Assisted Microphotometry. Isolation and Characterization of... 

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. 

Observations are made in metaphase cells arrested with a spindle inhibitor such as colchicine or colcemid to accumulate cells in a metaphase-like stage of mitosis (c-metaphase) before hypotonic treatment to enlarge cells and fixation with alcohol-acetic acid solution. Cells are then dispersed on to microscope slides and stained and slides are randomized, coded and analyzed for chromosome aberrations with high-power light microscopy. Details of the procedure are given in Dean and Danford (1984) and Preston et al. (1981, 1987). The UKEMS guidelines (Scott et al., 1990) recommend that all tests be repeated regardless of the outcome of the first test and... 

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... 

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

The localization of proteins and carbohydrates within cells and tissues with specific antibodies has long been proven to be a valuable technique. Immuno-localization procedures allow one to detect not only well-characterized cellular structures but also provide information about newly characterized proteins and carbohydrates. This chapter will review some of the advantages and drawbacks of common chemical fixation and permeabilization methods used for immuno-localization at the level of light microscopy. 

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. 

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