On Fri, 26 Jan 1996 06:26:06 -0800 M.J. Tobin wrote: snip... 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 Dear Mark The idea is that the intracellular environment alters the sensitivity of the probe so that calibrating inside the cell may be more appropriate. However, there is the problem as to whether or not you can accurately control the intracellular environment... For calcium perople use ionomycin or Bromo-A23187, but in our cardiac cells it has been hard for us to get Rmax (because the cells bleb) and Rmin (because the calcium takes forever to fall to zero). A good approach is to use a calibrating solution between Rmin and Rmax that does not alter the resting level when an ionophore is added. In my opinion, it is better to design experiments to look for changes rather than relying on an absolute calibration. The calibration can be given to allow others to compare their data but I wouldn't stake my reputation on an absolute calibration if I were you... To set the external solution pCa you can use EGTA buffers. Hope this helps, good luck Mark Cannell SGHMS