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