A Negative Stain for Electron Microscopic Tomography

Abstract

Negative staining can provide detailed, two-dimensional images of biological structures, but combining tomography with negative staining can provide three-dimensional images. Basic requirements for a negative stain for tomography are that the density and atomic number of the stain are optimal, and that the stain is not degraded with the intensive electron dose needed to collect a full set of tomographic images. A commercially available, tungsten-based stain, methylamine tungstate, appears to satisfy these prerequisites. Tomograms derived from multiple projections of EM images of the same structure yielded detailed images of single proteins on the surface of influenza A virus. Comparison of these images with published results from other methods served to evaluate this negative stain tomography. Images of surface renderings of the virus are a good fit to images derived from cryomicroscopy, as well as to the shapes of crystallized surface proteins. Thus, negative stain tomography provides realistic and detailed images of individual molecules in their normal setting on the surface of influenza A virus.

Direct visualization of CaMKII at postsynaptic densities by electron microscopy tomography

“…Measurements were limited to rings that were apparent in the xy plane, as definition was poorer in the xz and yz projections, although it was clear some rings were so tilted that they were not counted in the xy plane. Given the sizes of the areas sampled in the seven PSDs, there were 318 ± 36 (SEM) association domains per μm 2 of PSD, about half of the ≈600/μm 2 previously reported (Chen et al,2005) and about a fifth of the number estimated by Feng et al (2011).…”

C-terminal 'Tails' of Tubulin Revealed by Negative Stain EM Tomography

“…This work presents results obtained combining the electron microscopy tomography method with a recent method of sample preparation [1] which allows nanometer-scale resolution without averaging among many samples. Further, results have been improved using modern aberration-corrected microscopes and refined data analysis procedures.…”

“…When added in vitro to either dimeric tubulin or microtubules, Cr induces the formation of single protofilament rings that are highly monodisperse, composed of eight tubulin dimers resulting in a diameter of ~28 nm and thickness of ~4 nm [2]. Here we apply negative stain tomography [1] to Cr-tubulin rings. This technique, based on a beam stable organo-tungsten negative stain, when combined with proper data analysis, allows construction of tomograms that reveal nanometer scale details.…”

“…The sample has been embedded in a heavier than usual layer of stain so as to entirely embed the rings. A tungsten-based negative stain (NanoW, Nanoprobes) has been chosen [1] which, although destabilizing for microtubules, has no effect on these rings. The Cr-ring is a single protofilament, curved by Cr such that the external surface of the microtubule is the interior part of the ring.…”

Characterization of Hemagglutinin Antigens on Influenza Virus and within Vaccines Using Electron Microscopy

“…Phosphotungstic acid (PTA) is a widely used negative stain for influenza virus that produces a good contrast for viral glycoprotein spikes at neutral pH. Other negative stains used to visualize influenza include methylamine tungstate (NanoW) [ 62 ] and uranyl acetate [ 63 ]. Depending on the application, the low pH of uranyl acetate and related uranium-free stains may be of concern because conformational change of HA is triggered by low pH.…”