[FLIMfit-users] SymphoTime vs. FLIMfit

[Marko Šoštar]

Hi,
my name is Marko and I am a phd student on the Institute Ruđer Bošković in
Zagreb, Croatia.
I recently did some FLIM-FRET measurements where I tried to find out
FRET-ing population in a sample by fitting on the double-exponential model
N(t) = A1*exp(−t/t FRET) + A2*exp(−t/t 0). Ideally, I would measure donor
lifetime (t0) from donor only sample (mono-exponential decay), and then use
this as a fixed parameter in subsequent fitting procedures (with acceptor
present). And, that worked fine until recently, when I came across a sample
where very small change in donor lifetime (fixed parameter-I was playing
with manually changing it) would yield substantial difference in fitted A1
and A2, with equally good fit quality (chi-squared test). So, now I can't
decide which amplitudes to use. Does anybody have some advice how to
resolve this issue?
We use Leica confocal microscope paired with PicoQuant TCSPC system and
SyphoTime software for analysis. I should mention that I never measured IRF
(software offers to approximate it), so I'm not sure how this affects
fitting results. Also, I recently became aware of the FLIM fit software,
and I was wondering is there anyone experienced with both SymphoTime and
FLIM fit. What would be the better choice? Could this fitting issue be
resolved by using FLIM Fit (maybe it has some extra features for assessing
goodness of fit?)? Also, could you tell me what would be the best sample
(way) for measuring the IRF function (gold nanorods?- where to get them?).
I would greatly appreciate any advice.

Thank you and kind regards,
Marko Šoštar
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