Negative stain

Carmen A. Mannella cam14 at health.state.ny.us
Wed Sep 25 09:31:13 PDT 2002


Re Philip's comment:

In negative stain specimens, contrast is enhanced at the interfaces between
the biological material and metal cast.  The inherent low contrast of
internal details (like protein vs lipid domains in a membrane) is not
enhanced and, (in my experience) these details are not observed, presumably
due to the dominant contrast at the stain interface.  We see this in
comparisons of correlation averages of projection images of thin 2D
crystals of a pore protein (VDAC) in negative stains (UA or gold-glucose)
and in vitreous ice (Guo et al., 1995, J. Struct Biol. 114:41).   Both show
prominent water-filled pores but protein-lipid domains are visible only in
the frozen-hydrated and not the neg stained specimens.

This effect may be modulated by the density of stain used.  Maybe light
staining can be useful, e.g., by helping to  improve alignment at low dose
without greatly reducing visibility of internal stuctures.  I don't know
for sure but I think it's safe to say that neg stain generally yields
molecular outlines, including internal pores, pockets, crevasses, etc. that
can be penetrated by the stain.

Since most projects involve macromolecular assemblies in suspension (so
protein-lipid boundaries are not an issue) and don't go to high resolution
routinely (molecular outlines are the goal), I would heartily agree that
negative staining is usually worth exploring.

Carmen


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