In vivo calibration of fluorescent ion indicators:
 we have successfully applied the method of internal perfusion (via
patch pipette) with solutions of known calcium concentrations in
vascular smooth muscle several years ago (Wier & Blatter, Cell Calcium
12: 241-254, 1991). As has been pointed out by others, intracellular
binding of these dyes is one factor,among others, that might necessitate
in vivo calibrations for quatitative calcium measurements. Intracellular
binding of commonly used dyes can be substantial (Blatter& Wier, Biophys
Journal 58: 1491-1499, 1990).
 Lothar A. Blatter
 Dept. Physiology, Loyola University Chicago
 Maywood, IL 60153
 (708) 216-1182
 
> Most of the calibration methods published don't hold up very well because
> droplets don't behave at all like cells. Cells have complicated buffering
> systems and the Kd of the dyes can change a lot in some cellular
> environments. Punching holes in many cell types can be very slow. In my
> hands A23187 or ionomycin took 20+ minutes and I fell asleep by then.
> Then there's the issue of recoveries which I really like to show to prove
> thatI haven't done anything too weird to a cell. The only method that I
> ever thought worked was to perfuse a patch pipet with different amounts
> of Ca, which was worked out by Don O'malley. unfortunately, you need to
> be doing Patch loading.
>
>  Yu, et al, J. Neuroscience June94 14(6)3487
>  O'Malley, J. Neuroscience Oct 94 14(10)5741
>
>
>                                   Barry J Burbach