[FLIMfit-users] SymphoTime vs. FLIMfit

[Dunsby, Christopher W]

Dear Marko,

If you’re using multiphoton excitation, suitable gold nanorods can be purchased from Sigma and just dried on a coverglass, e.g.:



http://www.sigmaaldrich.com/catalog/product/aldrich/716820?lang=en&region=GB

This paper provides more information: doi: 10.1364/OE.19.013848.

RE the double exponential decay fitting, the decay parameters will be correlated so the interdependence that you see is normal. Using a high quality IRF and establishing a specific fitting protocol, e.g. by measuring the donor only lifetime from a separate sample and fixing it, should help you get reproducible and consistent results.

FLIMfit will let you do global analysis, e.g. if you have images then you can determine the lifetimes globally but the amplitudes for each pixel.

best regards,

Chris


From: FLIMfit-users [mailto:flimfit-users-bounces at lists.openmicroscopy.org.uk] On Behalf Of Marko Šoštar
Sent: 08 March 2017 13:16
To: flimfit-users at lists.openmicroscopy.org.uk
Subject: [FLIMfit-users] SymphoTime vs. FLIMfit

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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