Dear list members,

    We are trying to do FRET with CFP and YFP, which are the most common
combination used for this purpose. We have Chroma filters: Ex 436/20 BS
455DCLP and Em 480/40 for CFP; Ex 500/20 BS Q515LP and Em 520LP for YFP;
and Ex 436/20 BS 455DCLP and Em 520LP for FRET.
    I have several questions:
    1) Do you think this is the best combination? What about GFP and
DsRed?
    2) We have found a lot of bleedthrough of CFP in FRET filter set.
Does it usual? How do you solve it? Do you use other emission filter for
FRET and YFP?
    3) If it is not possible to avoid it, which is the kind of image
calculations do you usually develop? I suppose that it is very important
to have similar levels of signal with both constructions and set the
same exposure times when acquiring pictures in a CCD camera, isn't it?
Thank you very much in advance. I look forward to reading your comments.

Yours sincerely,
Carlos