[FLIMfit-users] FLIMfit and Time-Gated Data

[R.F. Laine]

Hi Susana,
I also use Erythrosin B in waterfor my IRF measurement.
Typically, to estimate the reference lifetime of Erythrosin B, I also 
acquire a sample with Rhodamine 6G and perform a monoexponential fit 
adding the reference lifetime as a fitted parameter.
I know that Rhodamine 6G lifetime is ~4ns and I can check whether the 
fit is performed correctly. You can do this on a central ROI of your 
image.

You'll need to estimate the IRF shift beforehand (also by fitting it), 
but beware that fitting both IRF shift and reference lifetime at the 
same time makes FLIMfit sometimes crash (fatal error) in my hands.

I hope this helps.

Cheers,

Romain




On 2017-01-24 15:36, Susana Silva wrote:
> Dear All
> 
> I am working with a Time-Gated FLIM system based on a LaVision
> intensified CCD camera capable of producing gates with widths ranging
> from 200ps to 1000ps. I am trying to used FLIMfit to fit the acquired
> data. Right now, I am trying to perform a monoexponential fit on a
> dataset acquired from a green chroma slide with an IRF acquired in
> fluorescence mode from a solution of erythrosine B in water (MiLiQi),
> but I have some issues regarding the quality of the fit and the
> reference lifetime I have to use in the IRF tab before fitting the
> data. I read somewhere in this mailing list that the reference
> lifetime should be estimated by a monoexponential tail fitting of the
> IRF curve. However, this advice was to TCSPC data and not Time-Gated.
> With Time-Gated IRF does the same procedure apply? If so, how can
> eliminate the gate effect when using large gate widths such as 1000ps?
> 
> 
> Best Regards,
> 
> Susana Silva
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-- 
Dr. Romain Laine, PhD in Biophotonics
Laser Analytics Group
Department of Chemical Engineering and Biotechnology
University of Cambridge
New Museums Site
Pembroke Street, Cambridge, CB2 3RA, UK
T: (+44)1223330133