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The following paper appeared in Proceedings of the Fifty-Second Annual Meeting, Microscopy Society of America; Bailey, G. W., and Garratt-Reed, A. J., Eds.; San Francisco Press, San Francisco, CA, 1994, pp. 132-133.

METHYLAMINE VANADATE (NANOVAN) NEGATIVE STAIN

James F. Hainfeld#, Daniel Safer*, Joseph S. Wall#, Martha Simon#, Beth Lin#, and Richard D. Powell**

#Biology Department, Brookhaven National Lab., Upton, NY 11973
*Department of Anatomy, The School of Medicine, University of Pennsylvania, Philadelphia, PA 19104
**Nanoprobes, Incorporated, 25 East Loop Road, Suite 124, Stony Brook, NY 11790

Uranyl and tungstate compounds have found favor as negative stains because of their high scattering power relative to biological molecules. However, other properties, such as specimen preservation, resistance to alterations or crystallization in the electron beam, and signal to noise (S/N) ratio, are also important. It may be that lower density materials may have advantages in these areas. A new negative stain, methylamine vanadate, CH3NH2VO3 ("NanoVan"1) offers a near physiological pH of 8, similar to phosphotungstate (pH 7) with much smoother background. It is also very stable in the electron beam with minimal granulation at a dose of 104 eI/nm2. The resolution obtainable with vanadate appears to be comparable to uranyl at low dose, but superior at higher dose where uranyl forms coarse grains (see Fig. 1). Problems with uranyl such as unwanted positive staining and the need for pH below 4 can be avoided. The lower contrast permits use of thicker stain embedment for better preservation and less flattening without excessive beam attenuation.

Contrast matching offers the possibility to locate components of a biological specimen by use of stains of different density. If comparable views can be recognized, the structures of interest will "disappear" in the contrast-matched image. Mixtures of glucose and gold thioglucose have been used for this application but suffer from radiation sensitivity of glucose and tendency of gold to form clumps at moderate dose. Mixtures of vanadate and borate are much more radiation resistant and suitable for contrast matching. Vanadate alone approximates the density of nucleic acid and borate is close to the density of protein. Complete embedment of the structure of interest is a requirement for interpretation and this can be obtained for objects as large as viruses due to the relatively low scattering power of vanadate/borate. No spurious effects due to positive staining have been observed to date with these stains. Various higher densities can be obtained by mixing withmethylamine tungstate2 ("Nano-W"2), a compatible high density stain which has a pH of 6.8.

Another useful property of methylamine vanadate is the clear visualization of 1.4 nm gold clusters ("Nanogold"1) in the presence of this stain. The gold core is much more dense than the stain and appears as a discrete spot with high contrast (Fig. 2). However, the undecagold cluster (Au11) does not give sufficient scattering to be observed with this stain. Either cluster can be observed in dark field STEM in otherwise unstained specimens, but with a resolution of fine features of 2-4 nm. In vanadate stain the resolution of small features is substantially improved. The gold clusters can be used either to locate specific features of interest in a protein matrix or, when attached to known sites, as fiducial marks to orient structures.

The vanadate/borate/tungstate negative stains therefore provide interesting and useful new tools for electron microscopists.

References

  1. Available from Nanoprobes, Inc., Stony Brook, NY, or E. F. Fullam.
  2. Oliver, R. M., Methods Enzym., 1973, p. 116.
  3. This work supported by NIH Biotechnology Resource Grant RR01777 and US Dept. of Energy, OHER.

[Figures] (72k)

Fig. 1. (a) Tobacco Mosaic Virus (TMV) negatively stained with 2 % uranyl acetate (A) and 1 % methylamine vanadate (NanoVan)(B) and imaged with a dose of 104 eI/nm2. Full width 128 nm.

Fig. 2. Side view of groEL (large arrow) labeled with 1.4 nm gold cluster (Nanogold, small arrow) imaged in methylamine vanadate. Note clear visibility of subunit structure and gold cluster. Full width 128 nm. Specimen kindly provided by A. Horwich, Yale University.

Thanks to the San Francisco Press for allowing us to reproduce this online.

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