Dear Pedro,
       Presumably you are loading your cells with the AM form
of fura2?  If it is being compartmentalized your cells may be bright
but your cytosolic calcium responses may be swamped by the
background from compartmentalized indicators.  Also, most of the
indicator inside cells (as least the "small" non-dextran linked indicators)
is usually bound to proteins which may increase its Kd by 4-fold or
more, making it harder to see small signals.  These problems may vary
with loading times, concentrations and also choice of indicator,
in idiosyncratic ways, so the best thing to do is try varying the
conditions.
     Fluo 3 has an inherently larger dynamic range than most other
calcium indicators, so its worth trying.  (Fluo 4 may be better but
I haven't tried it).  Also, have you tried looking at just one of your
fura wavelengths?  Ratioing often doesn't get you  "absolute"
calcium numbers because of other calibration problems, and you
may find one of your signals to be better than the ratio.  (Many
prominent fura papers published in recent years displayed all
their dynamic calcium data as single wavelength images).
Confocal should help, and the nuclear signals (if you can avoid
signal froom overlying cytoplasm) won't suffer from compartmentalization.

Stephen Baylor has published a number papers within the last 6 years
on the binding of indicators and specifically on the comparative
behavior of different AM indicators.

Good luck,
Don

Donald M. O'Malley, Ph.D.
Department of Biology
414 Mugar Hall
Northeastern University
Boston, MA 02115
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