Hello Fahad Mateen,

you don't write anything about the pecullarities of your experiment. But in case you are measuring in solution, try changing the concentration. FREt is negligible for donor-acceptor distances higher than about 10 nm. So if you rise concentration, FRET will appear rather abruptly at a value corresponding to that distance. If you do the same in a system without FRET, increase in acceptor emission will appear smoothly. But from your statement on 100 % spectral overlap I would guess it's FRET.

Good luck with your experiments 

Max

Thank you for your suggestion. 

I measured solid films. According to the formula, I found the foster critical radius as 2nm. Also, I changed the concentration. Acceptor:Donor -> 1:1, 1:2, 1:3. In all the cases, emission of donor completely diminished and acceptor emission rise.  I am curious that why donor emission is not appearing even at so high concentration?  

Can you tell me what donor and acceptor molecules you are using? And could you send me the excitation and emisson spectra of each and of the pair? Maybe I can find out something. By the way: 2 nm seems rather short for the Förster radius. And do you know anything about the orientation of the molecuels in your solid film?

Regads

Max 

FRET and Forster energy transfer are two different names for the same exact process. One is more popular with the biology/biochemistry community and one is more popular with the chemistry community.