First of all, thank you all for the helpful information regarding Fluo 3 and
Fura Red (and Calcium Green).  I was wondering if anyone has particular
comments on preparation of calcium standards to calibrate the microscope.  A
recent paper by Sanders et.al. (see end) describes lifetime imaging of pH in
cells using SNAFL-1, but the important point is that they describe calibration
of the microscope using labelled cells rather than solutions of known pH. (is
this common practice?)  They do this by loading the cells and making them leaky
to H+ ions.  Then by adjusting the pH of the surrounding medium, the pH inside
the cells is known.  They claim this is more accurate for calibrations.  Does
anybody know of a way of making cells 'leaky' to calcium ions and thus
measuring the reference solutions inside actual cells.
Any comments would be helpful
 
Mark Tobin
Daresbury Laboratory
 
 
Sanders R. et.al., (1995) Analytical Biochemistry 227, 302-308