[FLIMfit-users] FLIMfit and Time-Gated Data

[R.F. Laine]

Hi Ian,
Yes, this page describes the approach I mentioned. Susana, this is a 
great place to get acquainted with the method (and others actually).

The only difference is that it suggests measuring the IRF from scattered 
light from a cover slip, whereas I prefer measuring it from a short 
lifetime dye (same fluorescent filters as your measured data).
What I find surprising on this page is that I would not expect the 
scattered IRF mentioned there to have a reference lifetime (and should 
be used as a scatter) so it seems misleading to me.

On a side note, in general, the smaller the steps the better to 
describe/measure the IRF. The 25ps mentioned here relates to the fact 
that Kentech Fast delay boxes provide 25ps steps as the shortest steps 
possible.

Best,

Romain


On 2017-01-24 20:52, Munro, Ian wrote:
> Hi Romain
> 
> I know that our group uses IRFs with 25ps resolution for gated data,
> as described at
> http://docs.flimfit.org/advanced.html#creating-a-library-irf
> Can I ask how that relates to the process you describe?
> 
> Thanks and best wishes
> 
> Ian
> 
> 
> 
>> On 24 Jan 2017, at 16:25, R.F. Laine <rfl30 at cam.ac.uk> wrote:
>> 
>> 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
>>> _______________________________________________
>>> FLIMfit-users mailing list
>>> FLIMfit-users at lists.openmicroscopy.org.uk
>>> http://lists.openmicroscopy.org.uk/mailman/listinfo/flimfit-users
>> 
>> --
>> 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
>> _______________________________________________
>> FLIMfit-users mailing list
>> FLIMfit-users at lists.openmicroscopy.org.uk
>> http://lists.openmicroscopy.org.uk/mailman/listinfo/flimfit-users
> 
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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