Heavy metal negative staining, a now widely used routine technique to prepare biological material for imaging in the transmission electron microscope (TEM), was introduced almost 50 years ago (Brenner and Horne, 1959; for reviews, see Oliver, 1973; Horne, 1991). Suitable specimens may either be in solution or in suspension and thus include the whole spectrum of structural organization of biomolecules, ranging from monomeric and oligomeric proteins [e.g., RNA polymerase (Fig. 1a) or glutamine synthetase (Fig. 1b, bottom)], to protein polymers [e.g., cytoskeletal filaments or the multistranded helical cables of glutamine synthetase (Fig. 1b, top)], and eventually to large multicomponent supramolecular assemblies [e.g., nuclear pore complexes or bacteriophages (Fig. 1c)]. Compared to other electron microscopic techniques, negative staining is remarkably simple and can easily provide reliable structural information as quickly as 2min after preparing the specimen down to resolutions of 2.0nm (reviewed by Bremer et al., 1992) and below (Harris, 1999).
As shown schematically in Fig. 2a, a negatively stained specimen is embedded in a microcrystalline heavy atom salt replica that portrays its molecular architecture. Because heavy metal atoms (e.g., U, V, W, Au, Mo, Pt, Pb, Os) scatter electrons much more strongly than elements constituting the biomolecules (i.e., C, H, O, N, P, S), the contrast of negatively stained specimens (Fig. 2b) is much higher than that of unstained specimens and reversed (hence "negative" staining). In addition, the heavy metal salt replica stabilizes the specimen against collapse and distortion and substitutes, at least partially, for the aqueous environment typical for biomolecules. It also serves as a radiation protectant in the sense that it is more radiation resistant than biological matter (cf. Aebi et al., 1984; Bremer and Aebi, 1992).
II. MATERIALS AND INSTRUMENTATION
Negative staining requires only minor investment for instrumentation and supplies. For routine applications, the following items are needed.
The following solutions are required for negative staining.
A. Preparation of Negative Stain Solution
For the properties of various negative stains, along with references on their preparation, see Bremer et al. (1992). Here, the preparation of uranyl formate, one of the more delicate procedures, is described as an example:
(Mind, uranyl salts are radioactive, cf. p xxx)
Note: Uranyl formate may be difficult to purchase: Try, e.g., www.pfaltzandbauer.com (Pfaltz & Bauer Inc., Waterbury, CT 06708).
Our standard staining procedure for soluble proteins and their supramolecular assemblies is illustrated in Fig. 4. As might be appreciated, this procedure is quick, simple, and straightforward.
The quality of a negatively stained preparation (i.e., the interpretable structural detail captured on the EM screen, depicted on an electron micrograph, or monitored by a video or slow-scan camera attached to the EM) depends primarily on the penetration properties (e.g., size, charge, and hydrophobicity) of the heavy metal salt used for negative staining and on the physicochemical properties (e.g., solubility, charge, and hydrophobicity) of the specimen. Because some negative stains have a relatively acidic pH, whereas others are neutral or moderately basic and because they may be neutral, anionic, or cationic (for review, see Bremer et al., 1992), negative staining is quite versatile and can be tailored to specific needs.
When visualizing structures comprising components of different electron densities, e.g., colloidal gold-labeled proteins, the electron density of the heavy metal may become crucial. While 2- to 3-nm Au particles associated to bacteriophage T4 polyheads, i.e., aberrant tubular assemblies constituted exclusively of the major viral head protein gp23, are barely discernible from the uranyl acetate acetate-stained protein units (Fig. 5a), 3-nm Au-labled immunoglobulin G binding to the appropriate haemaglutinin antigen (Fig. 5b) and even 1- to 2-nm Au-labeled Fab fragments binding to haemaglutine assemblies (Fig. 5c), are still well discernible when stained with NaSiO4WO4, a less electron dense and intrinsically neutral negative stain (Baschong and Wrigley, 1990). A priori, the optimal stain for a particular specimen has to be chosen and evaluated by trial and error.
In our laboratory, starting in a first trial with an uranyl salt [we still favour uranyl salts and formate over acetate for it is slightly more stable in the electron beam (cf. Bremer et al., 1992)] or with a tungstate (e.g., NaPT, i.e., sodium phosphotungstate) proved successful with a wide variety of specimens, including cytoskeletal, membrane and soluble cytosolic proteins, and their supramolecular assemblies (for examples, see Aebi et al., 1983, 1984, 1988; Bremer et al., 1991, 1992, 1994; Hoenger and Aebi, 1996). Within this respect, it may be noteworthy that uranyl acetate can also act as a primary fixative: It was observed to stabilize bacteriophage T4 polyheads against dilutioninduced dissassembly in an aldehyde-like manner (Baschong et al., 1983; W. Baschong unpublished results) and was more recently documented to act as a primary fixative for skeleton muscle (Fassel and Graeser, 1997).
With tightening safety rules for the use of radioactive substancesmuranyl salts are radioactivem ordering, handling, and disposal of uranyl salts usually have specific guidelines (check with your safety responsible). In turn, the availability of uranyl salts may become more restricted, while novel readymade commercial methylamine-vanadate (Tracz et al., 1997) and methylamine-tungstate-based stains (Shayakhmetov et al., 2000) have been introduced (www.nanoprobes.com, Nanoprobes Inc., Yaphank, NY 11980-9710). As a consequence, nonradioactive metal salts such as tungstates, molybdates and vanadates may be given preference. The performance of such alternatives is best evaluated by trial and error, as illustrated in Figs. 6a-6e by staining bacteriophage T4 polyheads with uranyl formate (a) and prospective alternatives, i.e., with NH4MoO4 (b), NH4VO4 (c), Na2WO4 (d), and RhCl3 (e), and liposomes with methylamine vanadate (f).
The negative staining protocol described earlier is suitable for most specimens and stains. With delicate proteins, an additional washing step of the grid with water or sample buffer prior to applying the specimen may be included to wet the specimen support film and to minimize surface tension and/or denaturation of the specimen. The optimal sample concentration, which obviously has to be determined experimentally, should yield well-dispersed and evenly distributed particles adsorbed to the specimen support film. It is determined by the size, shape, and adsorption properties of the biomolecules or supramolecular assemblies under investigation, but also by the washing and staining regimen employed. For oligomers or polymers (e.g., such as filaments), crystalline, or paracrystalline specimens, a higher concentration than for individual biomolecules is required. As a rule of thumb, a few micrograms per milliliter for single particles (see Fig. 1a and bottom of Fig. 1b) and a few hundred micrograms per milliliter for supramolecular assemblies such as filaments (see top of Fig. 1b) or virus particles (see Fig. 1c) usually yield a reasonable particle density on the EM grid. The ionic conditions of the sample buffer and the presence of detergents, while strongly interfering with specimen adsorption, may have little effect on the quality of the negative stain replica surrounding the biomolecules, as long as a sufficient number of washing steps (usually two to six for most compounds) are employed prior to applying the negative stain solution to the specimen.
Heavy metal salts may not be inert but may interact physically or chemically with the specimen. Such sample-stain interactions may not explicitly produce "preparation artifacts" but sometimes unveil biologically significant information about the specimen going beyond purely structural aspects. For instance, human epidermal keratin filaments (cf. Aebi et al., 1983), after negative staining with uranyl formate (UF; pH 4.25), appear rather compact and featureless with a fairly uniform width (Fig. 7a). By contrast, when the same filaments are stained negatively with sodium phosphotungstate (NaPT; pH 7.0), they partially unravel and thereby unveil their protofibrillar substructure (Fig. 7b). This local unraveling becomes even more pronounced after washing the filaments briefly with 10 mM phosphate buffer (NaPi; pH 7.0) prior to staining them negatively with UF (Fig. 7c). Most likely, the inorganic phosphate (Pi) acts as a modulator of the lateral interaction of the protofilaments and/or protofibrils constituting the filament (cf. Aebi et al., 1983, 1988). In fact, a similar response to different negative stains has also been observed with F-actin filaments where inorganic phosphate appears to modulate the relative strength of the intersubunit bonds along and between the two long-pitch helical strands of actin subunits defining the filament (cf. Bremer et al., 1991, 1994; Bremer and Aebi, 1992).
A. Poor Specimen Adsorption
B. Patchy, Poor, or Bad Staining
C. "Bubbling" of Stain Upon Irradiation
D. Disintegration of Specimen During Preparation
E. Significant Background of Monomers and/or Small Oligomers with a Specimen Containing Supramolecular Assemblies
We thank Rosmarie Suetterlin and Daniel Mathys for their help. This work was supported by the Canton Basel-Stadt and the M.E. Müller Foundation of Switzerland)
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