Mark,
It is worth while to calibrate in the cell since not all of the dye may
be de-esterified to its active form (presuming that you load with an AM
ester rather than free dye), this affects the change of emission with
respect to calcium concentration.  Also difference of microenvironment
may influence the fluorescence.  Chused et al (Cytometry 8:
396-404[1987]) came up with a cocktail of nasties (including:ionomycin,
nigericin, potassium, azide, 2-deoxyglucose) that collapses the
transmembrane gradient with regard to calcium for mouse spleen cells,this
still doesn't allow Rmin to be measured, but that probably doesn't matter
too much since you're probably not interested in measuring calcium levels
lower than those encountered in resting cells.
Chused's method was developed for flow cytometry which lends itself less
to direct methods of measurement for Rmin, Sf2 and Sb2 than does
microscopy where measurements in solution are more readily made, but it
is still applicable.
 
 
Ray