We all know that the inner filter effect has a significant influence on the fluorescence emission spectra. The high concentration results in lower emission intensity. I wonder whether this effect is also able to affect the results of lifetime measurements if the concentration is very high.
Maybe you will receive second peak on life spectra with the same life-time?
What Amosov means is that you will see the lifetime of the fluorophore (normal), however, you might see a second lifetime that is due to quenching of the fluorophore (a decrease) or you might have a longer lifetime due to reabsorption (FRET).
At high concentrations fluorescence lifetimes may be observed to increase due to inner filter effects - this is described nicely by Birks (Photophysics of Organic Molecules p. 92), and if I may suggest a read of the following paper co-authored by myself: Photochem. Photobiol (1995) Vol 61 (4) 341-346. In this we show how the observed spectra and lifetimes of phthalocyanines change with concentration.
Can i ask if the dye you are looking at has a strong overlap between the absorption and emission spectra - ie a small Stokes shift and a high quantum yield? You might also find relevant articles if you look up 'radiative migration' which is the term Birks used to describe the effects of emission from the center of a sample being reabsorbed and re-emitted. Perhaps surprisingly under these conditions the emission still follows simple first order kinetics!
As Prof. Beeby states, the Birks treatment is the starting point, however when reabsorption is important, the decay departs from single exponential and also becomes emission wavelength dependent, see J. Chem. Phys. 90 (1989) 53, and Andrews and Demidov (eds.), Resonance Energy Transfer, 1999, chapter 3.
Andrew Beeby
"Hi Professor, could you please suggest how to correct the inner filter effect in TCSPC?"