On Thu, 25 Jan 1996 03:49:47 -0800 M.J. Tobin wrote:
 
 
> We have recently started using the dye mixture of Fluo 3 and Fura
Red to
> perform ratiometric measurements of calcium in living cells.  The
literature
> suggested these would work well as they show equal distribution
through the
> cells and little or no fading.  However I have been using viscous
solutions of
> the non cell permeant equivalent of each dye to check the alignment
of my
> instrument and have found that Fluo 3 bleaches very quickly.
> Has anybody else experienced this or has anyone a suggestion.
>
> Mark Tobin
> Fiona Lyng
>
Dear Mark
 
Fluo-3 has the best resistance to bleaching of any dye I have used. If
you are bleaching it you must be using very high laser intensities...
 
I assume you do have calcium present (or else the Fluo-3 doesn't
fluoresce very well). As a quick check of your laser intensity: Reduce
the light level by a factor of 3. Does the signal change? If not you
are in ground state depletion where the bleaching will occur at the
maximum rate possible. By the way, we use latex spheres for alignment
which bleach very slowly indeed.
Hope this helps.
 
Mark Cannell
 
SGHMS