Robert,

We have been using DM-NERF for pH measurements (intercellular space in
plants) and it works fine for ex488 & ex514 - dich 527 - em>585. It is
true that this dye doesn't have a true isosbestic point. However, the
emission caused by ex488 is less pH sensitive than that caused by ex514
thus ratio work is possible. We got quite steep and smooth calibartion
curves.

Peter

Dr Peter Kolesik ([log in to unmask])
Confocal Facility Manager
Waite Institute
University of Adelaide
Voice: +61_8_303 6523

On Wed, 20 Nov 1996, Robert England wrote:

> Does anyone have any experience of quantifying pH with DM-NERF (Molecular
> Probes D-2202, Set 21, p. 132) on a confocal microscope? Molecular Probes
> states that excitation ratios of 488/514 nm (or 450/500 nm for white light
> sources I guess) are suitable. Any comments to this in practical circumstances?
>
> As far as I can see from the excitation spectra of DM-NERF, the indicator
> doesn't have a true isosbestic point. Isn't a emission reading at an
> isosbestic point necessary to get a calcium-independent denominator for the
> ratio calculation?
>
> Robert England
>
> _____________R_o_b_e_r_t__E_n_g_l_a_n_d__________________________
>
>              E-mail:      [log in to unmask]
>              Tel:         +46 8 621 3400 x 3704
>              Fax:         +46 8 621 3470
>              Mobile:      +46 70557 3704
>
> _________________________________________________________________
>