Unconventional negative stains: Heavy metals are not required for negative staining

Salts of heavy metals (tungsten, uranium, and molybdenum) have long been used as negative stains for the transmission electron microscopy of protein molecules, supramolecular assemblies, and viruses, Negative staining still reveals details about protein structure only to around 25 إ (= 2.5 nm), most likely due to several major technical deficiencies in all traditional stain reagents. Experimental tests of unconventional compounds show that potassium aluminum sulfate, ammonium borate, and sodium tetraborate all dry into a glassy layer, and provide sufficient contrast to image the 86 إ lattice periods in thin crystals of catalase. Computer-generated power spectra of high-dose images indicate that sodium tetraborate can preserve periodic order in crystalline catalase to the same level (i.e., 28 إ) as the heavy metal negative stain, sodium silicotungstate. Sodium tetraborate also reveals the 40 إ wide central channel within tobacco mosaic virus, and the 25 إ thick protein shell of ferritin molecules. Images with these light atom compounds should be formed with a decreased electron beam exposure, in order to preclude the radiation-induced formation of voids within the dried stain. The results establish that heavy metal constituents are not necessary for a compound to function as a negative stain, and suggest that it should be possible to identify new reagents having the missing desired properties needed to obtain higher resolution imaging of protein structure with negative staining.