calibration of snarf

[David Brauer]

We have been using several different fluorescein based dyes to 
measure pH gradient within epidermal cells of roots from maize 
seedlings in situ. Treatment of the tissue with snarf-am under 
the appropriate conditions leads to a rather uniform labeling 
of both the vacuole and cytoplasm. Therefore, the snarf in 
these cells should be in pH environments ranging from pH 5 in 
the vacuole to almost pH 8 in to cytoplasm. We would like to 
be able to measure this pH gradient. The pH dependence graph 
of snarf in the Molecular Probes book makes one think it 
should be able to detect pH changes throughout this range. We 
have a calibration curve using a ratio of two excitation 
wavelengths of 420 and 475 nm at a constant emission 
wavelength of 580 + 30 nm.  However, this ratio varies only 
between pH 7 and 9. We have experimented with another 
calibration curve using a ratio intensities with  excitation 
and emission wavelengths of  420 nm and 580 nm compared to 
that obtained at exciation and emission wavelength of 480 and 
620 nm. With standard solutions, this ratio shows pH-dependent 
changes from pH 5 to 9 but the majority of the change is 
between pH 7 and 8. However, the calibration does not look as 
clean when one tries to do the calibration by clamping cells 
at various pH by various means.

Any suggestions would be appreciated.
 
I am also interested in getting know any other people in the 
Philadelphia PA area that are doing ratio imaging with plant 
cells.

Thank You for Your Reply in advance,
Dave Brauer
plant Physioogist
dbrauer at arserrc.gov