Lauran Oomen writes:
> As you all know, bleaching of fluorochromes during confocal laser
> scanning microscopy can be a very serious problem, especially when
> Z-series are to be made.
> I know of publicationsin which the effects of different anti-bleaching
> agents have been tested for conventional (total field illumination)
> fluorescence microscopy.
> I guess, however, that the characteristics of the anti-bleaching agents
> may be different when laser point scanning (high intensities, but for
> a short time and much more local) illumination is used.
> Since more and more anti-bleaching agents are coming on the market,
> I wondered whether anyone in this group has made a comparison of the
> effects of different anti-bleaching agents for use in confocal laser
> scanning microscopy. Currently we are using 'Vecta Shield', whith
> reasonable succes. The stuf is, however, also expensive.
>
We use p-phenylenediamine in buffered glycerol (GD Johnson et al., J. Immunol.
Meth. 55: 231-242, 1982).  It works well--fluorescein fades very slowly--but the
index of refraction is far from that of glass (or oil).  Thus you run into
spherical aberration in thick (e.g. 100 micron) sections.  For thinner sections
(e.g. 25 microns) it seems to be adequate, at least for some applications.
 
The real solution may be to use fluorophores such as bodipy, cyanine 5.18, or
the rhodamines, and mount them in something organic, like DPX.  Fading is low,
and the index of refraction is better--thus spherical aberration is lessened.
 
Martin Wessendorf