Dear Milena Mattes Cerveira ,

In my opinion, it should be possible to derive a fluorescence intensity ratio from microscopic images (comparable to a Fura-2 Ration imaging) but you should check your instrumentation (which is quite demanding).

You should either use an excitation of 488 and filter wheel for the emission filters, a dual camera setup with an appropriate filters (splitter) or if you are using fixed filter sets you would need two identical FITC filter sets and exchange the emission filter on that one for the red emission to the appropriate wave length (also check the beam splitter that it will work in conjunction with the red Emission filter).

Or you can use a psLSM and set the excitation to 488 and you are than reading out the emission in green and red simultaneously on two different detector .

You should also use CCCP as a positive control.

Kind regards and good luck

Soenke