阜新北站在哪个区:TEM Negative staining-九乡新闻网

a. Introduction

Fig. II.39. Schematic representation of a specimen particle completely embedded in a negative stain. (From Hayat and Miller, p.2)
For structural detail to be recorded in a micrograph, it must be faithfully preserved as the specimen is prepared and subsequently exposed to the vacuum and electron beam. The main purpose of negative-staining is to surround or embed the biological object in a suitable electron dense material which provides high contrast and good preservation (Fig. II.39). This method is capable of providing information about structural details often finer than those visible in thin sections, replicas, or shadowed specimens. In addition to the possibility of obtaining a spectacular enhancement of contrast, negative-staining has the advantage of speed and simplicity.
The potential value of negative stain microscopy derives from the relative ease with which specimens can be imaged at the molecular level of resolution. The technique has mainly been used to examine particulate (purified) specimens - e.g.. ribosomes, enzyme molecules, viruses, bacteriophages, microtubules, actin filaments, etc. at a resolution of 1.5-2.5 nm. This technique generally allows the shape, size, and the surface structure of the object to be studied as well as provide information about subunit stoichiometries and symmetry in oligomeric complexes. Any surface of the specimen accessible to water can potentially be stained, and thus, that part of the specimen will be imaged at high contrast.
b. Historical notes
Hall (1955) was the first to accidentally demonstrate the negative staining effect in a study in which particles were being positively-stained with phosphotungstic acid. Imperfectly washed particles were surrounded and embedded in the dried reagent and, instead of appearing dark on a light background, they were seen light on a dark background. Huxley (1957) independently noticed the same effect with tobacco mosaic virus. Brenner et al. (1959) also observed the same phenomenon with T2 bacteriophage and were the first to call it "negative staining".
c. Enhancing contrast of biological specimens
An object, whatever its chemical composition is, under normal conditions of TEM will barely be obvious (i.e. just noticeable contrast) in the electron image if the product of its thickness (in nm) and weight density (in g/cm3 is more than 10. Biological specimens have a density of about 1g/cm3, thus it is difficult in a micrograph to discern specimen features any smaller than 10 nm without some treatment to enhance its contrast. Positive staining involves treatment of the specimen with a chemical that increases the weight density, however, even intense staining that doubles the weight density would still only allow objects larger than 5 nm to be seen. When positively-stained, the object of interest appears in the image as an electron-opaque area against a relatively light background. Alternately, if the electron-transparent object is more or less completely embedded in a matrix of electron dense-material, and appears in reversed contrast as an electron-transparent area against a dark background, the object is said to be negatively-stained. Contrast enhancement by positive staining involves a direct interaction of a stain material with the protein. Negative staining does not require a stain-protein interaction, and denaturation of the molecule by staining is minimized, yet visualization to a limit of resolution of about 2.5 nm is easily realized.
d. Properties of an ideal negative stain
Each of the commonly used stains fails in some respect to meet the "ideal" specifications outlined below. An ideal stain should have:
- High density - Ability to protect specimen against dehydration effects- High solubility - Non-chemically reactive with the specimen- High melting and boiling points - Uniform spreading on the support film- Amorphous structure (i.e. structureless) when dry
High weight density to give high contrast
The density of most biological specimens ranges between 1.0 and 1.7 g/cm3. To increase the contrast of the specimen its mass thickness must be increased. The addition of non-specific stain to the specimen increases the specimen mass thickness, the effect being greater, the greater the mass density of the stain. Ideally the stain should be of uniform thickness and just sufficiently deep to completely engulf the specimen molecules. The "whole specimen" should be of limited thickness (10-50 nm). The resulting contrast of the specimen is much lower than that of the embedding amorphous film. In the photographic negative, the specimen appears dark against a light background. Hence the term "negative staining" which is not staining at all, but is embedding and is "negative" only in the sense that the biological material has less contrast than the surrounding material.
The density of most stains lies in the range 3.8-5.7g/cm3. The actual chemical composition of the stain has little effect on contrast. Note that the degree of contrast in the immediate vicinity of the object is determined by the density of the contrasting ion, not by the density of the contrasting substance as a whole. Heavy metal stains mostly cause elastic scattering of electrons, thus the main process of contrast formation is that of scattering contrast.
High solubility
The stain must be sufficiently soluble to allow enough to dissolve and come out of solution only in the final stages of drying. A relatively insoluble salt comes out of solution well before the drop finally dries and deposits as crystals without outlining or penetrating the specimen.
High melting and boiling points
The stain must be reasonably stable under the conditions to which the specimen is exposed in the microscope so it does not run or volatilize when heated by the electron beam. Energy transferred by inelastic scattering events produces radiation damage in the specimen. The rupture of chemical bonds with the formation of new structures and the ejection of materials from the irradiated area will eventually degrade the specimen. Transfer of energy to the specimen may also result in thermal degradation of the specimen and produce image drift due to thermal stresses in the specimen. Useful stains are highly resistant to such attack. Since the ultrastructural detail observable by negative staining is replicated by the stain, any degradation of the stain deposit during exposure to the beam will result in the loss of detail in the image.
Amorphous (non-crystalline) when dry
The drying of the stain and the specimen together with any buffer may produce artifacts, or contrast unrelated to the structure under study. Such "noise" degrades the quality of the image and may be of sufficient magnitude to obscure structural detail. To be most effective, the stain must deposit in an amorphous bed of uniform thickness and density to produce an artifact-free background against which the images of the specimen are viewed. A tendency to form microcrystals or other anomalies of structure or density in an otherwise homogeneous deposit will result in excessive "grain" effects in the slightly underfocused micrograph. Contrast from these effects may obscure the ultrastructural detail of the specimen.
If the molecular dimensions of the hydrated stain are the same or larger than the ultrastructure of the specimen, the visualization of that structural feature may be prohibited. Thus, some advantages may accrue from the use of stains of low molecular weight. Stains such as phospho-tungstate and silicotung state have dimensions of the order 0.8-0.9nm whereas uranyl stains are considerably smaller (0.4-0.5nm). This limits the possible resolution of specimen detail to values which are seldom if ever below 0.7 nm. Under normal conditions the resolution is limited to about 2 nm by inherent noise in the electron image. The highest resolution possible is obtained with negative stains such as uranyl formate, which are characterized by smaller size and hence increased penetrability. Less penetrating stains such as phosphotungstate are often used advantageously in systems where extensive penetration might obscure certain surface detail.
Non-reactive with the specimen
A most desirable, sometimes essential property of the stain is that it be chemically inert. An ideal stain is inert to the specimen as well as to the buffers, salts, metal ions, cofactors, or other reagents commonly used to maintain the integrity of the specimen structure. It is soluble and stable over a wide range of pH values. Any one stain may prove unsuitable for use with a particular specimen preparation by virtue of its reactivity with the specimen or the buffer solution. Uranyl salts are unstable at pH values >6 and are generally used below that pH. Uranyl ions form an insoluble compound with phosphate ions, thus, use of phosphate buffers in samples may lead to reaction between buffer and negative stain.
Partial disruption or changes in the tertiary structure of specimen molecules may occur with negative staining. This may prove advantageous or it may be necessary to chemically fix the specimen before staining to prevent disruption. Low concentrations (0.5-2%) of gluteraldehyde are sometimes used to fix the specimen before staining. To prevent inter-particle cross-links, the fixation is carried out for short periods (generally < 30 min) with extremely dilute samples. Another useful cross-linking agent is the bifunctional reagent dimethylsuberimidate which preserves positive charges at lysine residues in proteins, thus reducing conformational changes.
Protect against dehydration effects
In the final stages of drying, the dense stain ideally forms a uniformly thin, amorphous film in which the specimen is supported and preserved (Fig. II.39). The stain theoretically replaces the water in the interstices of the object until, in the dried specimen, all hydrated volumes are ideally filled with it. The enveloping stain then supports the specimen structure, if the deposit of stain is thicker than the embedded specimen. The specimen thus escapes exposure to surface tension forces and hopefully, retains its native morphology. A material of limited solubility may begin to precipitate before the last stages of drying and fail to engulf and protect the specimen. In general, those materials which make the best stains are very soluble.
Considerable changes in salt concentration and pH accompany drying of the specimens, but the stain helps protect the specimen from serious dehydration damage. When the specimen molecules are dried within a matrix of negative stain, surface tension forces are dissipated against the stain bed surface, thereby minimizing distortion of the specimen ultrastructure.
Uniform spreading on the support film
Excellent and uniform wetting of the support film surface is necessary to consistently achieve micrographs of good quality. A hydrophilic film surface enhances the tendency of the stain solution to spread and deposit the stain in an acceptably thin bed, free of artifacts (Fig. II.40). The support film may be made sufficiently hydrophilic either by glow discharging the grids or by treating the surface of the film first with a dilute (0.1 mg/ml) solution of cytochrome c or serum albumin before applying the specimen sample.
e. Additional notes about negative staining
1) Specimen purity
Negative stain microscopy is an excellent technique for assessing the purity of preparations and to appraise the efficiency of preparative procedures. Negative staining is of value in purification procedures, both for establishing a criterion of purity and for locating small amounts of a particular specimen particle in a fractionation scheme.
2) One- verses two-sided staining
With negative staining, sometimes only the top or bottom of a particle is contrasted with the stain. This usually occurs if the stain layer is very thin. In thicker layers of stain, particles will generally stain on both sides. There seems to be no way to reproducibly obtain one- or two-sided images for most specimens. Also, it is often difficult to ascertain the degree to which stain embeds both sides of a particle. The use of holey films (see Sec. II.A.3.g.1) provides some control over achieving more uniform staining of particulate specimens.

Fig. II.40. Schematic representation of the effect of the support film and the specimen surface on the meniscus of the stain before drying. (a) Hydrophilic specimen on hydrophilic support film. The adhesiveness of the stain to both the specimen surface and the support film is almost equal; it is greater than the cohesiveness of the stain. (b) Hydrophilic specimen on hydrophobic film. The adhesiveness of the stain to the specimen surface is stronger than the adhesiveness to the film, but the adhesiveness of the stain to the film is less than the cohesiveness of the stain. (c) Hydrophobic specimen on hydrophilic film. The adhesiveness of the stain to the film is stronger than the adhesive-ness to the specimen surface. The adhesiveness of the stain to the specimen surface is less than the cohesiveness of the stain. (d) Hydrophobic specimen on hydrophobic film. The adhesiveness of the stain to the specimen surface is greater than stain cohesiveness, but the stain cohesiveness is less than the adhesiveness of the stain to the film. Alternatively, the adhesiveness of the stain to the specimen surface is equal to the adhesiveness to the film, but the adhesiveness to the film is stronger than the cohesiveness of the stain. (From Hayat and Miller, p.23)
3) Positive vs. negative staining
Since most biological specimens have charged groups exposed to the surrounding aqueous solvent, and the stain molecules are ions, there will almost always be some degree of positive staining (Fig. II.41). For example, phosphotungstate ions are negatively charged and they can be used most effectively as a negative stain only for specimens raised above their isoelectric point so they do not attract the contrasting ions. Uranyl acetate at pH 4.5 is used as a negative stain with specimens that are positively charged below pH 5. With the anionic stains such as the phosphotungstates, positive staining is expected in the region of low (acidic) pH values because of the powerful interaction between positive charges in the specimen and the negatively charged tungstate ions.

Fig. II.41. The effects produced by (a) positive staining, (b) negative staining, and (c) metal shadowing on a fragment of collagen lying on a supporting membrane. The overall effect of banding is clearly shown by all three methods, but each supplies certain information which is lacking in the others. Shadowing shows clearly the granular nature of the carbon support film. (From Meek 1st ed., p.466)
4) Maintenance of biological structure/function
It has been found that some viruses are still infectious after mixing and spraying with phosphotungstate and certain enzymes remain active even after they have dried in droplets of stain and are subsequently rehydrated.
5) Reliability of images
It is always important, especially with unfamiliar specimens, to try a variety of conditions (stain, pH, temperature, concentration of specimen, stain and buffer, etc.) when preparing specimens for microscopy using negative staining techniques. Quite often, under varied conditions, different features of a specimen will be enhanced, and either complementary or perhaps even contradictory information may be obtained. If, for example, a variety of stains are used and similar staining patterns are obtained, then it is likely that the features revealed are consistent with genuine specimen morphology.
f. Common negative stains
The following table lists some chemicals used as negative stains along with some of their properties:

阜新北站在哪个区:TEM Negative staining

II.A.3. Negative-Staining [原文地址]