Scanning microscopy. Supplement最新文献

Two critical factors in the preparation of metal films on biological specimens are the type of metal used and the potentially damaging effects of radiant energy from the hot metal source. The excessive heating of surfaces is a major limitation to the replication of heat-sensitive aqueous specimens with refractory metals such as tungsten and tantalum, although these metals are known to form smaller grains and thinner films than the more commonly used platinum/carbon deposited under similar conditions. We describe here an electron gun designed for the evaporation of pure tantalum; surface heating is reduced through intermittent deposition controlled by varying the open/closed intervals of a fast shutter that operates in ultra-high vacuum. The effectiveness of the shutter was evaluated with a thin thermocouple in place of the specimen. The composition of the replicas was determined by x-ray microanalysis and by direct observation of the initial melting and subsequent evaporation of the tantalum bead supported on a tungsten rod that remained unchanged during the evaporation. The quality of the tantalum replicas was demonstrated with freeze-fracture replicas of reconstituted proteoliposomes and native membrane vesicles. With shutter intervals of 0.5 sec open and 1.0 sec closed, the surface heating was reduced enough to prevent unintentional etching and to preserve small pits complementary to protein particles in hydrophobic membrane surfaces and in ice.

A computer assisted morphometric method has been elaborated to quantify synaptic vesicles evidenced by means of the Zinc-Iodide-Osmium (ZIO) staining procedure in nerve endings of a very discrete area of the cerebellar granular layer: the glomerulus. The following parameters were calculated directly on electron microscopic negatives of 4.67 microns 2 of surface terminal area: number of vesicles per unit area (Na), and per unit volume (Nv), volume density (Vv), average diameter (d) and average volume of the single vesicle (V). Ultrastructural changes taking place at nerve endings also cover synaptic vesicles, thus quantitative studies regarding vesicle population at synaptic regions can be correlated to functional changes occurring in the process of chemical transmission and reflect the plasticity of synaptic junctional zones. Although this histochemical staining method generally is referred to as unspecific, after comparing our data with the available literature reports, we propose that ZIO-positive vesicles could have a physiological significance. These ZIO-positive organelles could take part in the intraterminal homeostatic control of Ca++ ions.

There is no single method which would provide an unambiguous image of all types of biological macromolecules. The choice of method depends largely on the size and properties of the macromolecule. Obviously small molecules are best visualized by negative staining, the problems appear with negative staining of larger structures. Here, the uncertainty about which part of the complex is actually stained (top or bottom) makes correct interpretation difficult. Shadowing techniques have the advantage of both visualizing the surface and also delineating the whole macromolecule, but suffer from lower resolution due to the graininess of the metal. However, they are superior to negative staining for the visualization of thin linear macromolecules. The next series of problems includes the interaction of macromolecules with supporting films, glass coverslips or mica, which can be hydrophobic, hydrophilic or charged and these properties can influence the orientation of the molecules. Surface tension forces during air-drying must also be considered. We have used a variety of preparative techniques in our studies of biological macromolecules: (a) negative staining; (b) air-drying from ethanol; (c) glycerol-spraying; (d) adsorption freeze-drying; (e) monolayer freeze-etching. These methods have been tested on small viruses, water soluble proteins (ribosomes, F-actin, microtubules) and transmembrane proteins requiring the presence of detergents (sarcoplasmic reticulum ATPase, fibronectin receptor). We find that freeze-drying is the most reliable and easy method for molecules that withstand distilled water; freeze-etching can be successfully applied to transmembrane proteins (even in the presence of detergents or salt); the glycerol-spray technique provides an excellent alternative to the cryotechniques in particular for studies of single linear molecules.

The physical events of the cryoultramicrotomy at the level of organelles and macromolecules are not completely understood. The extent to which tissue is either cut by the edge of the knife or fractured ahead of the knife is one such event. This issue of cryofracturing versus cryosectioning during cryoultramicrotomy has been examined in quick frozen, uncryoprotected rat liver. Cryosectioned specimens were freeze-substituted and edge-on views of the sectioned surface were examined in TEM. In tissue regions showing no obvious ice crystals, fracturing was rare. Regions with less adequate freezing however had numerous fractured structures. These results indicate that high quality freezing promotes sectioning over fracturing and thus works to eliminate this serious artifact.

The high packaging ratio of DNA in both interphase nuclei and metaphase chromosomes presents great difficulties to our understanding of the three dimensional organisation of processes such as DNA replication and transcription in the nucleus. Although the higher order structure of DNA, in terms of the way it is organised into the unit fibre of chromatin has received much attention over the last decade, the highest levels of packaging of chromatin in both nuclei and chromosomes have hardly begun to be elucidated. Much of the difficulty in investigating fibre organisation with conventional methods is the inherent two dimensional nature of sectioned or spread material in the transmission microscope. Three dimensional imaging from the SEM has, until recently, been limited by the available resolution. Our own previous studies of chromosome structure have shown that a combination of 'in lens' imaging combined with the high signal generation imparted by osmium impregnation have been adequate to routinely visualise chromatin fibre organisation in metaphase chromosomes, and the changes that occur as a result of a variety of banding techniques. More recent experiments using the same techniques on interphase nuclei extracted from a variety of tissue culture cells have indicated that Scanning electron microscopy of nuclei is a potentially useful technique for studying chromatin organisation, which may be made more accessible by a variety of biochemical extraction methods.

The resolution currently available in both transmission and scanning electron microscopes is theoretically adequate to visualize the organization of the cytoskeleton at the supramolecular and macromolecular levels. However, achieving this resolution in practice requires that the methods used to prepare the specimens both preserve the structures of interest and render them visible for observation in the microscope without obscuring or altering them. In this paper we discuss our own and others efforts to develop methods to overcome several problems associated with preparing whole mounts of cytoskeletons for observation by electron microscopy. These problems include: controlling the degree to which cellular components are extracted; the effects of osmium tetroxide on the cytoskeleton; controlling and recognizing shrinkage and drying artifacts; the choice of a method of visualization; deposition of grain-free ultrathin films of metal; and interpreting the results. The standard procedure which we currently use consists of the following steps: growing cells on carbon-stabilized Formvar-coated gold electron microscope grids; extracting in 0.5% Triton X-100 detergent in a microtubule stabilizing buffer; postfixing in 2.5% glutaraldehyde in stabilizing buffer; freeze-drying; magnetron sputter-coating with 1.5 nm of tungsten; and observation by TEM, SEM, or STEM. Cytoskeletons prepared in this manner contain over 100 polypeptides and are composed of a complex three dimensional meshwork of clean, uniform filaments, the smallest of which are 7 nm in diameter. A structure resembling the microtrabecular lattice is present only if the cells are prefixed with a relatively long bifunctional protein crosslinking reagent prior to extraction with detergent.

Colloidal gold can be produced in sizes ranging from 1.0nm to 150nm. All sizes of gold can be conjugated, principally by hydrophobic bonding, to a variety of molecules including ligands, enzymes and antibodies, as well as lectins and polysaccharides. The activity of most of these biological molecules is retained on conjugation with gold particles irregardless of size range, although the ratio of protein surface area to gold particle surface area varies widely depending on particle and protein size. We have employed low voltage high resolution scanning electron microscopy to compare, microscopically, the shapes of biological molecules unbound, bound to very small (3nm) gold particles, and bound to larger (18nm-30nm) gold particles. When very small gold particles are conjugated to large protein molecules, several particles bind along the length of each molecule, while smaller protein molecules often wrap around a single small gold particle. With larger gold particles, several biological molecules bind to a single gold particle. In addition, the shape of protein molecules bound to larger gold particles differs from that of molecules bound to small gold particles.

Frog sartorius and semitendinosus muscles are quick-frozen either in the resting state or during contraction by means of a LN2 cooled falling copper block. The frozen specimens are freeze-substituted (acetone + OsO4 + uranyl acetate) in a REICHERT JUNG CS auto and either embedded in Spurr's resin and polymerised at a high temperature (60 degrees C) or embedded and polymerised in the Lowicryls K4M, K11M or HM23 at low temperatures (below -30 degrees C). Excellent morphological results are obtained when freeze-substitution, embedding and polymerisation are all carried out below -50 degrees C. Muscles in which a major portion of cellular K+ ions has been replaced by electron dense Cs+ or Tl+ ions are also cryofixed at rest or during contraction, freeze-substituted in pure acetone for 1 week at -80 degrees C and polymerised in K11M at -60 degrees C. A characteristic uneven distribution of the electron dense ions--known from earlier published control experiments--can be observed in sections of resting muscles. Electrically stimulated muscles show ion redistribution. It is concluded that freeze-substitution and low temperature embedding of quick-frozen contracting muscle may be used to investigate changes of ultrastructure, redistribution of cellular water and intracellular movements of mobile ions during muscle contraction.

High pressure freezing permits the successful cryoimmobilization of thick biological specimens (up to approx. 500 microns). A very high yield of adequately frozen specimens, in which no segregation patterns due to ice crystal formation is apparent after freeze-substitution or freeze-fracturing, is obtained with suspensions of microorganisms as well as plant and animal tissue. This very high yield is attributed to an optimized transfer of pressure and cold to the biological specimen. This is achieved by replacement of extraspecimen water or buffer by 1-hexadecene, a chemically inert, hydrophobic paraffin oil of low viscosity and low surface tension.

This paper describes an image analysis technique for the assessment of changes in chromatin organization in ultrathin tissue sections. Transmission electron micrographs of tissue sections were analyzed by a SEM-IPS image processing unit (Kontron) at a total magnification of 7,500. The boundaries of each nucleus and each nucleolus were defined interactively, and the grey level threshold between heterochromatin and euchromatin was determined. The grey level distribution and the total area of each nucleus was measured. In addition, the total area, number, and individual areas of heterochromatin particles and nuclei were measured. Based on these measurements, a number of different variables have been defined, and several of these have proved to discriminate between normal and malignant cells.