Abstract
Negative stain electron microscopy (EM) allows relatively simple and quick observation of macromolecules and macromolecular complexes through the use of contrast enhancing stain reagent. Although limited in resolution to a maximum of ~18 - 20 Å, negative stain EM is useful for a variety of biological problems and also provides a rapid means of assessing samples for cryo-electron microscopy (cryo-EM). The negative stain workflow is straightforward method; the sample is adsorbed onto a substrate, then a stain is applied, blotted, and dried to produce a thin layer of electron dense stain in which the particles are embedded. Individual samples can, however, behave in markedly different ways under varying staining conditions. This has led to the development of a large variety of substrate preparation techniques, negative staining reagents, and grid washing and blotting techniques. Determining the most appropriate technique for each individual sample must be done on a case-by-case basis and a microscopist must have access to a variety of different techniques to achieve the highest-quality negative stain results. Detailed protocols for two different substrate preparation methods and three different blotting techniques are provided, and an example of a sample that shows markedly different results depending on the method used is shown. In addition, the preparation of some common negative staining reagents, and two novel Lanthanide-based stains, is described with discussion regarding the use of each.
Highlights
Despite recent attention to the resolution revolution resulting from significant advances in cryo-electron microscopy[1], negative stain EM remains a powerful technique and a crucial component of electron microscopists' toolbox
All of the staining reagents tested produced negative staining to some degree, with uranyl formate (UF) yielding the samples with the greatest contrast and sharpest, most detailed particles
The larger grain size of Thulium Acetate (TmAc) becomes apparent at high magnification, when Tobacco Mosaic Virus (TMV) particles were stained with 1% TmAc the ~23 Å repeat of the TMV particle[17] was still clearly visible by eye and as a meridional layer line in the Fourier transform of the raw image
Summary
Despite recent attention to the resolution revolution resulting from significant advances in cryo-electron microscopy[1] (cryo-EM), negative stain EM remains a powerful technique and a crucial component of electron microscopists' toolbox. Negative staining still remains the best method for rapid assessment of a sample before optimizing cryo-grid conditions[2]. The high contrast and speed of grid preparation of negative stained samples makes it ideal for assessing sample purity, concentration, heterogeneity, and conformational flexibility[3]. A high relative contrast is produced between the background and the particle of interest, with the particle being less electron dense than the surrounding stain[8]. The particles appear as light areas because of their low electron scattering power relative to the dense surrounding stain, which scatters the electrons more and appears darker. Substructural features of particles can be deduced from the detailed examination of resultant images as stain will penetrate into any crevice and produce irregular contrast detail[9]
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