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Hi Guy,

On Fri, 13 Mar 2015 13:10:05 +0000, Guy Cox <[log in to unmask]> 
wrote:
>I've never tried to do this, but could one not use standard volumes of a standard
>dye concentration, for example in a multiwell slide?

I like that approach, but I think that the diffusion of the dye in a liquid combined 
with the relatively small imaging area will mean this will take a *long* time to 
bleach. And some dyes have different properties when actually bound to a protein 
or antibody.

I've measured photobleaching of dyes by embedding them in agarose or gelatin 
and sandwiched that between a coverslip and slide. You could add spacer 
microbeads to the mixture if you want to maintain a certain thickness. If you 
normalize by brightness/absorption, the exact concentration of the dye shouldn't 
matter, but keep it relatively low and as similar sample-to-sample to avoid issues 
such as self-quenching, dimerization, reabsorption, etc.

You could also consider something immobile like a simple immunofluorescence 
sample. Of course, you'd have to remake that often, and if your staining protocol 
isn't well worked out, you might get significantly different brightnesses between 
samples. But it might be a starting place.

If you're just trying to compare microscope systems and not dyes, there's always 
Tetraspeck beads.