> Subject: calcium measurements
> I would like to generate a concensus about the best ways to measure
> calcium in the confocal. I would like to propose a flow sheet of choices
> and then let everyone rip it to shreds (this is what friends are for...).
>
> Here is my proposed flow chart for dye selection.....
>
> 1. INDO-1 is the first line choice of calcium-sensitive dye because of
> dual-emission ratios. INDO-1/AM is a crummy esterase substrate, but if
> you get enough calcium-sensitive fluorescence in your cells it will work.
> Major hassle is calcium-INsensitive fluorescence from the remaining
> INDO-1/AM (sometimes reduced by post incubation without dye). INDO-1 also
> requires a UV laser. When loading is poor or you have no UV laser, move
> on to other choices. Only exception is if you REALLY need quantitative
> results, because as you leave INDO-1 you move into more qualitative
> methods: in this case invest time in optimizing dye loading.
 
It also bleaches PDQ.
>
> 2. FLUO-3 and FURA-Red (or some such combination) is the next best choice.
> This method of dual dye loading can be semi-quantitative over
> short time intervals. Each days loading is different, so must normalize
> in some way to compare results day-to-day (eg. to ratio in resting cells
> before "stimulation"). Must prove that dye loss/photobleaching is
> negligible (or similar between probes) over the time course of study. A
> major advantage of these dyes is that there is little or no fluorescence
> from AM forms, and the higher exciatation and emission spectra keep
> autofluorescence to a minimum. In the confocal, differences in
> compartmentalization between dyes may not destroy results IF dye does not
> shuttle between compartments during stimulation (i.e. ratios at any
> cellular site should be valid, but don't compare results in the nucleus
> versus results in cytoplasm unless you can prove [dye] is about the same in
> each compartment).
 
Dual dye loading with the AM methods is *really* risky for 'accurate'
calibration. Better to inject the dyes...
>
> 3. FLUO-3 alone is the simplest method for identifying sites of calcium
> mobilization. Use the method when this is your only goal: it is only
> qualitative because there is no ability to do ratioing in any form. Good
> for recognition of calcium mobilization (since fluorescence increases it
> can not be ascribed to common artifacts of dye loss/bleach). Fluo-3 is a
> good choice because the 488 line (and common FITC filters) make it
> accessible in most confocals.
 
There is an ability to ratio the data. You can express it as F/Frest. If you
know roughly what calcium Frest is due to then the record can be calibrated.
(See Cannell et al., Biophys J. 67 1942-1956). This method relies on the fact
that Fmin~0 so you only need to know Fmax or the calcium associated with some
particular level of F. In my experience this method works suprisingly well in a
confocal due to the constant volume of the voxel from which you are recording.
On the other hand, I always treat calibaration with some scepticism... I
recommend always doing experiments that look for relative changes rather than
calibrating absolute levels...
 
That's my two cents worth for now.
 
Mark Cannell
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--
Mark B. Cannell
Reader in Biophysics
SGHMS