On Mon, 7 Nov 1994, Geoff Hyde wrote:
 
> Thanks very much for the tip on the Biophysical Journal article on SNARF
> (vol 66, pp 942-952). One point of correction for other readers of your
> message- the emission curves in this paper show that the isosbestic point
> for excitation at 568nm is 585nm, not 610nm. This latter wavelength is the
> isosbestic point for excitation at 488nm.
>
> Geoff Hyde
> [log in to unmask]
> Biology Dept,
> York University, Ontario.
>
 
One other point to pay attention at, not only with SNARF but with every
intracellular fluorescent dye, is that their spectra change when the dyes
are in the cell.
 
The published graphs in the above paper were for in vitro calibration.
We have done the calibration in vivo with rat cardiac myocytes and the
isosbestic point was at 592.
 
The tricky point though is that at 568 excitation, the short emission
wavelengths do not change much with pH and the usable fluorescence in the
band between 585 and 590, quite narrow even for the best filters.
 
 
Stamatis Pagakis
University Laboratory of Physiology
University of Oxford, Parks Rd.                FAX:  +44 (0)865 272469
Oxford OX1 3PT,   United Kingdom               Tel:  +44 (0)865 272434