欧洲什么贵:TEM Negative staining
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II.A.3. Negative-Staining [原文地址]
Ammonium molybdate' NH4Mo7O24o4H2O 44 2.5 Sodium phosphotungstate Na3PO4o12WO3 ? 3.8 Sodium tungstate
Oliver (1973) reports that methyl phosphotungstates and methylamine tungstate may be used as negative stains. They have the primary advantage of a greater tendency to wet the support film surface, spreading over sufficiently large areas of relatively thin stain bed of good uniformity. They appear to be stable over a wide range of pH values (ranging from pH 4 to 9.5).
g. Negative Staining procedures
Techniques for the preparation of negative stain specimens are simple and direct. The essential aim of the procedure is to embed the specimen in a uniformly thin deposit of stain. Resolution of molecular features is only accomplished at the stain-specimen boundary where there is maximum contrast. This result is only achieved if the deposition of buffer salts or other materials with densities less than the stain at that boundary are severely limited; otherwise the specimen molecules will be imaged at low resolution and will appear as nondescript blobs.
The specimen sample is usually applied directly to the surface of the support film where a population of specimen particles becomes adsorbed. Attachment of the molecules is usually secure enough that they are not removed by subsequent rinsing and staining operations which do remove most of the buffer salts. Since different specimens often have different affinities for the particular support film being used, some measure of control over how much specimen attaches to the film may be effected by adjustment of the specimen and buffer concentrations, adsorption time, etc. Appropriate conditions must be established by experiment for each new specimen. For protein solutions, typical concentrations range between 50-500 ug/ml and adsorption times from as little as 1-5 seconds to several minutes. Stains are usually applied in a range of concentration from 0.25-4.0%. Adjustment of stain concentration provides some control over the thickness of the deposit.
It does NOT follow that a procedure that is successful with one type of specimen is also suitable for another, so various modifications should always be tried until good contrast and spreading conditions are achieved.
There are two common procedures for preparing negatively-stained specimens on EM grids:
Adhesion (drop) method (Figs. II.42 and II.43)
A droplet of specimen is placed on the surface of the grid support film, making sure it sufficiently wets the surface. After an appropriate time interval, excess specimen is wicked away by touching a piece of filter paper to the edge of the grid surface. Without letting the grid dry, a droplet of rinse or stain solution is applied to the grid. Rinsing is necessary if the specimen preparation contains high concentrations of buffer salts or other solutes which may interfere with deposition of stain. The nature of an appropriate rinse depends on the conditions that the specimen can tolerate. Many viruses, for example, can withstand rinsing with distilled water. In some instances the stain solution can itself act as a suitable rinse. After rinsing and staining, excess fluid is wicked from the grid, leaving a thin aqueous film on the surface which is left to dry, usually in air.
Fig. II.42. Preparation of a specimen from particles in aqueous suspension. (From Hall, p.290) Fig. II.43. Washing a specimen. (From Hall, p.290)
The specimen can also be applied to the support film by floating the grid on top of a droplet of the specimen solution. The grid is then transferred to droplets of rinse and stain solutions and then dried as before.
An additional variation of the usual adhesion method is to apply the sample to a holey support film in the same way as is done on regular films. The sample dries in a thin layer of stain stretched out over the holes, thereby giving maximum contrast since there is no plastic and/or carbon support. Also, the stain tends to be more evenly distributed around the particle although the particle often undergoes distortions (shrinkage and flattening) due to the surface tension forces created as the layer of stain dries. The stain layer also has a tendency to break either before or after it is exposed to the electron beam. The layer of stain can be stabilized with a thin layer of evaporated carbon. Another advantage of this technique over the usual method is that, if small enough, the specimen particles will be randomly oriented in the stain layer. On regular support films, particles often settle on the surface of the film in one or a few preferred orientations, thus limiting the possible views of the specimen.
Spray droplet technique(Fig. II.44)
The normal adhesion method of preparing a particulate suspension may lead to erroneous conclusions about the relative proportion of particles since different particles are likely to have different affinities for the substrate. Also the microscopist may select fields attractive to the eye but which are not representative. The only reliable way of preparing specimens without introducing a bias is to dry a drop of the original sample in its entirety. Non-volatile salts and buffers must be removed by centrifugation and washing or by dialysis so they don't obscure the particles under study or alter the structure when the salt concentrates in the last stages of drying. The entire residue from the drop must be examined, thus it is necessary to obtain very small drops. The suspension is atomized as a fine mist and the droplets are allowed to impinge on the substrate.
Fig. II.44 A simple hand-held nebulizer from which the sample (s) is sprayed onto mica (m). (From Willison and Rowe, p.69)
The spray droplet technique is particularly useful for examining specimens that adsorb so poorly to the support film that application and removal of rinsing and stain solutions also removes the specimen. Appropriate volumes of the specimen and stain solutions are mixed and sprayed in small droplets onto a wetable support surface. If the solution itself has the propensity to wet and spread over the surface, uniformly thin deposits of negatively-stained specimen result. The resultant aqueous film will be of uniform depth, and the mass of stain deposited per unit area of support film tends to be constant. When the specimen appears to have dried, some water may still be present in the stain bed, and a rearrangement of the stain deposit could result from its rapid vaporization if the specimen is suddenly placed in the vacuum of the microscope. Thus, the specimen is usually allowed to dry (sometimes over a desiccant) for at least 10 minutes.
Drying of the aqueous film proceeds from the edges, the central area covered by the droplet being last to dry. Minor solutes tend to be held in solution until the last stages of drying and are deposited in highest concentrations in the central area. As a result, the specimen in this area ordinarily is of inferior quality.
Fig. II.45. Drop pattern. The small particles are tomato bushy stunt virus, and the large spheres are polystyrene latex particles 2,600 A in diameter. The wedge-shaped sector has been magnified and superimposed. (From Hall, p.359)
Note that, using the adhesion drop method, there may be preferential adherence of particles so relative particle distribution counts cannot be made. A major advantage of the spray technique over the adhesion method is that preferential adherence to the grid of one type of particle over another type of particle in a mixture cannot occur. Thus, this is the method of choice in quantitative studies where relative concentrations of particles in the sample need to be determined. By knowing the volume of the original drop (from adding known concentrations of polystyrene spheres) a count of the number of particles in a drop pattern provides immediately the number of particles per unit volume (Fig. II.45: Note that this figure shows a metal-shadowed specimen). This, together with the mass per unit volume obtained by weighing the dried residue from a measured volume can be used to calculate a value for the mean molecular weight of the particles.
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