Copied from e-mail conversation.
Dear David
That’s good news. A lot of the features of FLIMfit are detailed here :
https://www.openmicroscopy.org/site/pro ... s.pdf/view
However, more roughly:
Delta function irfs allow a "quick & dirty" way of looking at data in the absence of an experimental irf.
They simply assume a ‘perfect’ measurement system & therefore that the excitation pulse is coincident with the first non-zero data point.
(because a perfect system will detect no light before the excitation).
Try excluding all the data before the peak using the Data-> Time Min option 7 then ‘Fit Selected Decay’.
A pixel from the file that that you sent me fitted like this, using a Delta function irf is shown below.
The ability to shift the irf in time is provided for situations where it is known that the measured irf is shifted in this way.
Usually this is due to the difference in wavelength between the lift used to measure the irf & the data & the different detector response
at these 2 wavelengths.
The ability to include this shift in the fit ( where it is not known in advance )is provided by “Fit IRF Shift”.
The “Fit Reference” option is only relevant if you are using a “Reference IRF” (i.e. a Fluorophore with a known lifetime) as opposed to a scatterer IRF
where the lifetime is known to be extremely short .
I’m not entirely sure exactly what you want to do re ROIs but I would suggest having a look at the segmentation manager.
However, It isn’t currently possible to display more than one decay at the same time.
Regards
Ian
<Screen Shot 2014-02-26 at 09.42.24.png>
On 25 Feb 2014, at 16:39, David Lleres wrote:
Dear Ian,
Thanks for your email and sorry for my silence.All is fine.
No worry regarding the last issue with FLIMfit and bio-formats.
Since last time i've been trapped in understanding all parameters and how to use properly FLIMfit software. So far, I still have a couple issues with the IRF and especially to understand some parameters such as IRF background, IRF shift, set delta fct IRF. Also in the lifetime window I'm still doubting if I need to leave "Fit reference" and "Fit IRF shift" in fixed or fitted. Perhaps all is related to an old IRF data that I'm using. I will record a new one this week with guy from microscopy facility and will try again.
Nevertheless , I succeed to fit the decay and lifetime from past acquisitions and lifetime value obtained seems in the good agreement to what I had using spcimage, and all in one it is nicely working.
In this view, I would like also to know if there are some possibilities to select more than 1 ROI in the same image and have the decay lifetime displayed and associated for each ROI ? Same question, when I load for example 2 images from one data set, then when I draw a ROI in one data, the same region is automatically select in the other data file even if it didn't contain any information.
Regarding your question, unfortunately I don't have a .sdt file containing multi-channel data acquired using measurement_mode 13. Sorry for that.
Many thanks for your help and I'll let you know my progress.
Best wishes,
David
David Lleres, PhD
IGMM (Institute of Molecular Genetics of Montpellier)[
