Dear Johannes,

you are right that the ratio algorithm increases noise, however by using
fura-2 as a ratiometric dye you gain a lot of freedom and robustness
against the many other strong artefacts which can occur with the single
wavelength dyes as movement, volume changes, uneven loading, different
bleaching, non similar behaviuor of two different dyes used for ratioing
and....

So I would think fura-2 has for sure a lot of advances even if it means
UV in the confocal. But as we have shown with the 'old' Zeiss LSM 410
(Pfluegers Archiv 433, 653-663) it can be done and now with the ZEISS
LSM 510 (we do it routinely and published several papers for example see
  JBC 1998, Vol 273) it works very good and the newer LEICAs TCS
SP2 should do it too. In my mimd in the confocal it is less critically
to use fura-2 in difficult preparations than
one wavelength dyes. Still we work with dyes from both groups as the one
wavelength dyes named before by other members of the confocal user group
can have advantages under some conditions too.

So our general way when starting with a new object is: try several dyes
including fura-2 if you have the confocal for UV and then you will see
which works best under your conditions. There are quite often surprises
about dyes being sequestered or transported out of the cells, which can
not be foreseen. So I agree fully to the arguments of Michael, Robert
and Christian.

Good luck Hermann for your experiments.

Roland


___________________________

Nitschke, Roland Dr.
Institute of Biology I
Life Imaging Center
Albert-Ludwigs-University Freiburg
Hauptstr.1

D-79104 Freiburg
Germany
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phone: 49-761-2032934 or 2902
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