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Hi Jan
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<div>That’s great. </div>
<div>Simply select a region using the ROI tools (Circle,square polygon) top-left</div>
<div>then right-click on the displayed decay & export as a .csv.</div>
<div>The .csv file can then be loaded as an IRF.</div>
<div><br>
</div>
<div>Just FYI to load a region as a background select in the same way then Background->Use Selected Region as Time Varying Background.</div>
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<div>Best </div>
<div><br>
</div>
<div>Ian</div>
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<div>On 18 Sep 2014, at 20:41, Borst, Jan Willem <<a href="mailto:janwillem.borst@wur.nl">janwillem.borst@wur.nl</a>> wrote:</div>
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Hi Ian, Paul<br>
<br>
Thanks for updating this topic<br>
<br>
I am able to perform tail fitting, and get expected values.<br>
I am now playing with setting and will try to get lifetime heat map if possible.<br>
We measure in plant cells which contain very fast BG fluorescence, is it possible to select a region and define that as IRF?<br>
<br>
Thanks<br>
<br>
JW<br>
<br>
<br>
Dr Jan Willem Borst<br>
assistant professor<br>
AFSG, Laboratory of Biochemistry<br>
Microspectroscopy Centre<br>
Wageningen University<br>
Visiting address: Dreijenlaan 3, building 312, 6703 HA Wageningen, the Netherlands<br>
T: +31317483724/+31654930887<br>
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I:<span class="Apple-converted-space"> </span><a href="http://www.biochemistry.wur.nl/">www.biochemistry.wur.nl</a><<a href="http://www.biochemistry.wur.nl/">http://www.biochemistry.wur.nl</a>>,<span class="Apple-converted-space"> </span><a href="http://www.mscwu.wur.nl/">www.mscwu.wur.nl</a><<a href="http://www.mscwu.wur.nl/">http://www.mscwu.wur.nl</a>><br>
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<br>
On 18 Sep 2014, at 19:35, Munro, Ian <<a href="mailto:i.munro@imperial.ac.uk">i.munro@imperial.ac.uk</a><<a href="mailto:i.munro@imperial.ac.uk">mailto:i.munro@imperial.ac.uk</a>>> wrote:<br>
<br>
Hi Paul<br>
<br>
Thanks for that. It’s good to see a community starting to build up.<br>
<br>
A couple of general points to note.<br>
<br>
You don’t always need to select an ROI. It can .however, be useful to use this to combine data from a region to give a low-noise decay<br>
on which you can then try things like different models & backgrounds by doing ‘Fit Selected Decay’ before going on to fit the whole image/dataset.<br>
<br>
<br>
It’s not always good to assume the level before the peak is background. Depending on the rep rate of the laser & the lifetime of your sample<br>
some fluorescence from the previous pulse may still be visible.<br>
By default FLIMfit allows for this in the fit, if you set the 'Rep .rate' box correctly or this feature can be disabled using ‘Pulse train Correction’ on the ’Advanced’ tab.<br>
<br>
<br>
Time varying background. This option is provided for where there is exactly that.<br>
This could arise from, in our case, fluorescence from a plastic sample holder appearing in the data or autofluorescence from cells.<br>
The best approach is to measure a background witth no sample but everything else in place: Culture medium etc as appropriate.<br>
<br>
Also note the 'Bg is afterpulsing' option on the IRF tab. (Not all TCSPC systems suffer from after afterpulsing)<br>
<br>
<br>
<br>
Re your last question:<br>
<br>
FLIMfit will include the TVB in it's fitting model so you won’t se an effect on the data only on the fitted lifetime.<br>
<br>
Oh and one other point , for pixel-by-pixel fitting using TCSPC we suggest changing the Algorithm to ‘Maximum Likelihood’ using the ‘Advanced’ tab.<br>
This is optimal for Poisson noise (although somewhat harder & therefore slower computationally.<br>
<br>
All the best<br>
<br>
Ian<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
On 18 Sep 2014, at 14:00, Paul Thomas (SCI) <<a href="mailto:P.Thomas@uea.ac.uk">P.Thomas@uea.ac.uk</a><<a href="mailto:P.Thomas@uea.ac.uk">mailto:P.Thomas@uea.ac.uk</a>>> wrote:<br>
<br>
Hi Jan and Ian,<br>
<br>
Here is my basic protocol for analysis using FLIMfit - this is REALLY basic as I am very much a beginner:<br>
<br>
FLIMfit basic fitting:<br>
<br>
1) Load FLIM data<br>
2) Draw ROI on area to be analysed<br>
3) In palettes below, under "Data" choose Smoothing = 5x5 (B&H 2) and set Integrated Min to appropriate value (Threshold level for counts) [Note: If NOT using an IRF set Time Min to start time of the fluor peak.]<br>
4) In Background palette choose Background = Single value and iteratively try various numbers until the first part of the fit (pre-peak) is close to 0<br>
5) At bottom under "Lifetime", choose Global fitting = "Pixel-wise"<br>
6) From IRF menu choose "Load IRF", and open IRF file - if necessary choose "Estimate IRF shift" from the same menu (stop iterations when Function value reaches a minimum)<br>
7) At the very bottom choose "Fit Dataset" - data is shown in Parameter palette beneath the Decay window; satisfactory fit is indicated by low Chi2 value (<1.2) - try shifting IRF again, or changing other parameters if fit not good<br>
8) Tau map is obtained in Image palette by checking tau_1 Dis. box, and adjusting Min and Max values<br>
<br>
A question for Ian - I was advised to use "Time varying background", but I haven't been able to get this to work, ther doesn't seem to be any shift in the baseline when I choose an ROI in the background of my image, and no indication that background has been
subtracted. Any clues as to where I'm going wrong?<br>
<br>
Thanks,<br>
Paul.<br>
______________________________________<br>
Dr. Paul Thomas, Manager,<br>
The Henry Wellcome Laboratory for Cell Imaging,<br>
Faculty of Science,<br>
University of East Anglia,<br>
Norwich Research Park,<br>
Norwich,<br>
NR4 7TJ,<br>
United Kingdom.<br>
e-mail:<span class="Apple-converted-space"> </span><a href="mailto:p.thomas@uea.ac.uk">p.thomas@uea.ac.uk</a><<a href="mailto:p.thomas@uea.ac.uk">mailto:p.thomas@uea.ac.uk</a>><br>
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Personal web-page:<span class="Apple-converted-space"> </span><a href="https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab/people">https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab/people</a><br>
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<br>
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-----Original Message-----<br>
From:<span class="Apple-converted-space"> </span><a href="mailto:ome-users-bounces@lists.openmicroscopy.org.uk">ome-users-bounces@lists.openmicroscopy.org.uk</a><<a href="mailto:ome-users-bounces@lists.openmicroscopy.org.uk">mailto:ome-users-bounces@lists.openmicroscopy.org.uk</a>>
[mailto:ome-users-<br>
<a href="mailto:bounces@lists.openmicroscopy.org.uk">bounces@lists.openmicroscopy.org.uk</a><<a href="mailto:bounces@lists.openmicroscopy.org.uk">mailto:bounces@lists.openmicroscopy.org.uk</a>>] On Behalf Of Munro, Ian<br>
Sent: Thursday, September 18, 2014 1:55 PM<br>
To: Borst, Jan Willem;<span class="Apple-converted-space"> </span><a href="mailto:ome-users@lists.openmicroscopy.org.uk">ome-users@lists.openmicroscopy.org.uk</a><<a href="mailto:ome-users@lists.openmicroscopy.org.uk">mailto:ome-users@lists.openmicroscopy.org.uk</a>><br>
Subject: Re: [ome-users] [FLIMfit-users] FLIMfit is not fitting B-H data nor PQ<br>
data<br>
<br>
Hi Jan<br>
<br>
No problem/. Whenever you have the time.<br>
<br>
Best<br>
<br>
Ian<br>
<br>
<br>
On 18 Sep 2014, at 13:49, Borst, Jan Willem <<a href="mailto:janwillem.borst@wur.nl">janwillem.borst@wur.nl</a><<a href="mailto:janwillem.borst@wur.nl">mailto:janwillem.borst@wur.nl</a>>> wrote:<br>
<br>
Hi Ian<br>
<br>
Would be great, but I have to postpone it to next week as I am on<br>
travelling coming days and have to work on a proposal ..<br>
<br>
Thanks.<br>
<br>
JW<br>
On 18 Sep 2014, at 14:44, Munro, Ian <<a href="mailto:i.munro@imperial.ac.uk">i.munro@imperial.ac.uk</a><<a href="mailto:i.munro@imperial.ac.uk">mailto:i.munro@imperial.ac.uk</a>>> wrote:<br>
<br>
Hi Jan<br>
<br>
I simply meant that the SNR of the fitted lifetime is much lower using the<br>
tail-fitting approach as you discard a lot of the light.<br>
<br>
Would it be possible to see one of your data sets & associated irf ?<br>
<br>
Perhaps we could then work out what the problem is.<br>
<br>
Best<br>
<br>
Ian<br>
<br>
<br>
On 18 Sep 2014, at 13:36, Borst, Jan Willem <<a href="mailto:janwillem.borst@wur.nl">janwillem.borst@wur.nl</a><<a href="mailto:janwillem.borst@wur.nl">mailto:janwillem.borst@wur.nl</a>>><br>
wrote:<br>
<br>
Hi Ian<br>
<br>
We have IRF measured and have tried to use it and have tried it without.<br>
No fitting possible<br>
S/N is pretty good especially if you use bin option.<br>
<br>
Talk to you later<br>
<br>
JW<br>
On 18 Sep 2014, at 14:09, Munro, Ian <<a href="mailto:i.munro@imperial.ac.uk">i.munro@imperial.ac.uk</a><<a href="mailto:i.munro@imperial.ac.uk">mailto:i.munro@imperial.ac.uk</a>>> wrote:<br>
<br>
Dear Jan<br>
<br>
I guess the first question is " do you have a measured irf ? ".<br>
If not , then you may , if your data is truly mono-exponential and<br>
there are enough photons,. be able to get a good lifetime by using the<br>
tail- fitting approach that I describe here:<br>
<br>
<a href="http://lists.openmicroscopy.org.uk/pipermail/flimfit-users/2014-Sep">http://lists.openmicroscopy.org.uk/pipermail/flimfit-users/2014-Sep</a><br>
tember/000005.html<br>
<br>
For anything other than pure mono-exponential decays and in order<br>
to maximise your SNR we strongly suggest measuring an irf along with<br>
each experiment.<br>
<br>
Some information about FLIMfit and irfs is available at<br>
<a href="https://github.com/openmicroscopy/Imperial-FLIMfit/wiki/GUI-walkthr">https://github.com/openmicroscopy/Imperial-FLIMfit/wiki/GUI-walkthr</a><br>
ough (about half-way doen the page.)<br>
<br>
All the best.<br>
<br>
Ian<br>
<br>
<br>
<br>
<br>
<br>
On 18 Sep 2014, at 12:48, Borst, Jan Willem <<a href="mailto:janwillem.borst@wur.nl">janwillem.borst@wur.nl</a>><br>
wrote:<br>
<br>
Dear all<br>
<br>
We are trying to use FLIMfit 4.7.0 for analysing FLIM data.<br>
We are able to load B-H data as well as PQ data (bin format) and obtain<br>
the intensity image.<br>
Selection of a pixel displays the decay curve but not good fit is<br>
obtained.<br>
<br>
Can someone maybe post guidelines which settings should be used?<br>
There is FLIMfit documentation but that does not give a protocol of the<br>
steps to take.<br>
<br>
All info is welcome.<br>
<br>
Best<br>
<br>
JW<br>
<br>
<br>
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