Photoactivable Ca/Voltage probes

Enric Claverol etc97r at ecs.soton.ac.uk
Tue Mar 28 07:29:35 EST 2000


Dear all,

I need a hand regarding photoactivable Ca/Voltage sensitive probes.  I
am looking
for a Ca/Voltage sensitive probe with memory. I would like to find a
photoactivable
probe (ideally ratiometric) which  ""freezes"" into its current state
when illuminated
(phtoactivated).

In other words, I am interested in measuring the Ca concentration at a
particular
point in time and from many location in the tissue (in particular at
several dephs in the tissue).
Because the number of cells from which I want to do the measurement
is high, I can not use a time changing probe. Ideally, I would like to
have a "freezing" probe
which retains a particular conformation (which must be  Ca or voltage
dependent) upond
illumination with the activation wavelength. After illumination, I would

scan the whole
tissue measuring the Ca concentration that was in the cell at the point
in time of interest.

There may be an alternative to having such a complex probe. May be
combining two
simpler probes.

In another variation of the experiment, I would like to have an in-vitro

preparation and apply
the "freezing" Ca sensitive probe. After some time, I would activate the

Ca probe which would
freeze into one of two fluorescense states (A which emits wavelength A
or B which emits wavelength
B). The probability of freezeing in state A or state B should be
dependent on Ca concentration (or voltage).
After the activation flash, I would fixate the tissue (with a protocol
which conserves fluorescense
from the probe) and  microtome-slice the tissue. Then, I would extract
3D info about the Ca concentration
at the time of the activation flash.

I will appreciate any suggestions and apologies for this long email.


Enric T. Claverol
University of Southampton
U.K.







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