[ome-users] [FLIMfit-users] FLIMfit is not fitting B-H data nor PQ data

Paul Thomas (SCI) P.Thomas at uea.ac.uk
Fri Sep 19 08:34:52 BST 2014 

Thanks Ian,

I've changed my protocol to:

1)	Load FLIM data
2)	Draw ROI on background of image, from Background menu choose "Use Selected Region as Time Varying Background"
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.]
4)	In Background palette choose Background = TV Image
5)	At bottom under "Lifetime", choose Global fitting = "Pixel-wise"
6)	Under Stray, choose TVB = 1
7)	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)
8)	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
9)	Tau map is obtained in Image palette by checking tau_1 Dis. box, and adjusting Min and Max values


Is this correct (in particular steps related to Background - 2, 4 & 6?

Regards,
Paul.
______________________________________
Dr. Paul Thomas, Manager,
The Henry Wellcome Laboratory for Cell Imaging,
Faculty of Science,
University of East Anglia,
Norwich Research Park,
Norwich,
NR4 7TJ,
United Kingdom.
e-mail: p.thomas at uea.ac.uk
Tel: +44-1603-592196
Fax: +44-1603-592250
Imaging web-site: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab
Personal web-page: https://www.uea.ac.uk/biological-sciences/research/facilities/henry-wellcome-lab/people


UK Top 15 (14th in the Guardian University Guide 2015; 15th in the Complete University Guide 2015)
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>-----Original Message-----
>From: Borst, Jan Willem [mailto:janwillem.borst at wur.nl]
>Sent: Friday, September 19, 2014 6:54 AM
>To: Munro, Ian
>Cc: Paul Thomas (SCI); ome-users at lists.openmicroscopy.org.uk
>Subject: Re: [ome-users] [FLIMfit-users] FLIMfit is not fitting B-H data nor PQ
>data
>
>Thanks will give it a try
>
>Sent from my iPad
>
>On 18 Sep 2014, at 22:14, Munro, Ian
><i.munro at imperial.ac.uk<mailto:i.munro at imperial.ac.uk>> wrote:
>
>Hi Jan
>
>That's great.
>Simply select a region using the ROI tools (Circle,square polygon) top-left then
>right-click on the displayed decay & export as a .csv.
>The .csv file can then be loaded as an IRF.
>
>Just FYI to load a region as a background select in the same way then
>Background->Use Selected Region as Time Varying Background.
>
>Best
>
>Ian
>
>
>On 18 Sep 2014, at 20:41, Borst, Jan Willem
><janwillem.borst at wur.nl<mailto:janwillem.borst at wur.nl>> wrote:
>
>Hi Ian, Paul
>
>Thanks for updating this topic
>
>I am able to perform tail fitting, and get expected values.
>I am now playing with setting and will try to get lifetime heat map if possible.
>We measure in plant cells which contain very fast BG fluorescence, is it
>possible to select a region and define that as IRF?
>
>Thanks
>
>JW
>
>
>Dr Jan Willem Borst
>assistant professor
>AFSG, Laboratory of Biochemistry
>Microspectroscopy Centre
>Wageningen University
>Visiting address: Dreijenlaan 3, building 312, 6703 HA Wageningen, the
>Netherlands
>T: +31317483724/+31654930887
>F: +31317484801
>E:
>JanWillem.Borst at wur.nl<mailto:JanWillem.Borst at wur.nl><mailto:JanWillem.
>Borst at wur.nl>
>I:
>www.biochemistry.wur.nl<http://www.biochemistry.wur.nl/><http://www.
>biochemistry.wur.nl<http://www.biochemistry.wur.nl/>>,
>www.mscwu.wur.nl<http://www.mscwu.wur.nl/><http://www.mscwu.wur.
>nl<http://www.mscwu.wur.nl/>>
>www.wageningenur.nl/en/Disclaimer.htm<http://www.wageningenur.nl/en
>/Disclaimer.htm><http://www.wageningenur.nl/en/Disclaimer.htm>
>
>On 18 Sep 2014, at 19:35, Munro, Ian
><i.munro at imperial.ac.uk<mailto:i.munro at imperial.ac.uk><mailto:i.munro at i
>mperial.ac.uk>> wrote:
>
>Hi Paul
>
>Thanks for that. It's good to see a community starting to build up.
>
>A couple of  general points to note.
>
>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 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.
>
>
>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 some
>fluorescence  from the previous pulse may still be visible.
>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.
>
>
>Time varying background. This option is provided for where there is exactly
>that.
>This could arise from, in our case, fluorescence from a plastic sample  holder
>appearing in the data or autofluorescence from cells.
>The best approach is to measure a background witth no sample but
>everything else in place: Culture medium etc as appropriate.
>
>Also note the 'Bg is afterpulsing' option on the IRF tab. (Not all TCSPC systems
>suffer from after afterpulsing)
>
>
>
>Re your last question:
>
>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.
>
>Oh and one other point , for pixel-by-pixel fitting using TCSPC we suggest
>changing the Algorithm to 'Maximum Likelihood' using the 'Advanced' tab.
>This is optimal for Poisson noise (although somewhat harder & therefore
>slower computationally.
>
>All the best
>
>Ian
>
>
>
>
>
>
>
>
>On 18 Sep 2014, at 14:00, Paul Thomas (SCI)
><P.Thomas at uea.ac.uk<mailto:P.Thomas at uea.ac.uk><mailto:P.Thomas at uea
>.ac.uk>> wrote:
>
>Hi Jan and Ian,
>
>Here is my basic protocol for analysis using FLIMfit - this is REALLY basic as I am
>very much a beginner:
>
>FLIMfit basic fitting:
>
>1) Load FLIM data
>2) Draw ROI on area to be analysed
>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.]
>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
>5) At bottom under "Lifetime", choose Global fitting = "Pixel-wise"
>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)
>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
>8) Tau map is obtained in Image palette by checking tau_1 Dis. box, and
>adjusting Min and Max values
>
>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?
>
>Thanks,
>Paul.
>______________________________________
>Dr. Paul Thomas, Manager,
>The Henry Wellcome Laboratory for Cell Imaging, Faculty of Science,
>University of East Anglia, Norwich Research Park, Norwich,
>NR4 7TJ,
>United Kingdom.
>e-mail:
>p.thomas at uea.ac.uk<mailto:p.thomas at uea.ac.uk><mailto:p.thomas at uea.a
>c.uk>
>Tel: +44-1603-592196
>Fax: +44-1603-592250
>Imaging web-site: https://www.uea.ac.uk/biological-
>sciences/research/facilities/henry-wellcome-lab
>Personal web-page: https://www.uea.ac.uk/biological-
>sciences/research/facilities/henry-wellcome-lab/people
>
>
>UK Top 15 (14th in the Guardian University Guide 2015; 15th in the Complete
>University Guide 2015) UK Top 3 for Student Experience (Times Higher
>Education Student Experience Survey 2014) World top 1% (Times Higher
>Education World Rankings 2013-14) World Top 100 (Leiden Ranking 2014)
>
>
>
>This email is confidential and may be privileged. If you are not the intended
>recipient please accept my apologies; please do not disclose, copy or
>distribute information in this email or take any action in reliance on its
>contents: to do so is strictly prohibited and may be unlawful. Please inform me
>that this message has gone astray before deleting it. Thank you for your co-
>operation.
>
>-----Original Message-----
>From: ome-users-bounces at lists.openmicroscopy.org.uk<mailto:ome-users-
>bounces at lists.openmicroscopy.org.uk><mailto:ome-users-
>bounces at lists.openmicroscopy.org.uk> [mailto:ome-users-
>bounces at lists.openmicroscopy.org.uk<mailto:bounces at lists.openmicroscop
>y.org.uk><mailto:bounces at lists.openmicroscopy.org.uk>] On Behalf Of
>Munro, Ian
>Sent: Thursday, September 18, 2014 1:55 PM
>To: Borst, Jan Willem; ome-users at lists.openmicroscopy.org.uk<mailto:ome-
>users at lists.openmicroscopy.org.uk><mailto:ome-
>users at lists.openmicroscopy.org.uk>
>Subject: Re: [ome-users] [FLIMfit-users] FLIMfit is not fitting B-H data nor PQ
>data
>
>Hi Jan
>
>No problem/. Whenever you have the time.
>
>Best
>
>Ian
>
>
>On 18 Sep 2014, at 13:49, Borst, Jan Willem
><janwillem.borst at wur.nl<mailto:janwillem.borst at wur.nl><mailto:janwillem.
>borst at wur.nl>> wrote:
>
>Hi Ian
>
>Would be great, but I have to postpone it to next week as I am on travelling
>coming days and have to work on a proposal ..
>
>Thanks.
>
>JW
>On 18 Sep 2014, at 14:44, Munro, Ian
><i.munro at imperial.ac.uk<mailto:i.munro at imperial.ac.uk><mailto:i.munro at i
>mperial.ac.uk>> wrote:
>
>Hi Jan
>
>I simply meant that the SNR of the fitted lifetime is much lower using the tail-
>fitting approach as you discard a lot of the light.
>
>Would it be possible to see one of your data sets & associated irf ?
>
>Perhaps we could then work out what the problem is.
>
>Best
>
>Ian
>
>
>On 18 Sep 2014, at 13:36, Borst, Jan Willem
><janwillem.borst at wur.nl<mailto:janwillem.borst at wur.nl><mailto:janwillem.
>borst at wur.nl>>
>wrote:
>
>Hi Ian
>
>We have IRF measured and have tried to use it and have tried it without.
>No fitting possible
>S/N is pretty good especially if you use bin option.
>
>Talk to you later
>
>JW
>On 18 Sep 2014, at 14:09, Munro, Ian
><i.munro at imperial.ac.uk<mailto:i.munro at imperial.ac.uk><mailto:i.munro at i
>mperial.ac.uk>> wrote:
>
>Dear Jan
>
>I guess the first question is " do you have a measured irf ? ".
>If not , then you may , if your data is truly mono-exponential and there are
>enough photons,. be able to  get a good lifetime by using the
>tail- fitting approach that I describe here:
>
>http://lists.openmicroscopy.org.uk/pipermail/flimfit-users/2014-Sep
>tember/000005.html
>
>For anything other than pure mono-exponential decays and  in order to
>maximise your SNR we strongly suggest measuring an irf along with each
>experiment.
>
>Some information about FLIMfit and irfs is available at
>https://github.com/openmicroscopy/Imperial-FLIMfit/wiki/GUI-walkthr
>ough (about half-way doen the page.)
>
>All the best.
>
>Ian
>
>
>
>
>
>On 18 Sep 2014, at 12:48, Borst, Jan Willem
><janwillem.borst at wur.nl<mailto:janwillem.borst at wur.nl>>
>wrote:
>
>Dear all
>
>We are trying to use FLIMfit 4.7.0 for analysing FLIM data.
>We are able to load B-H data as well as PQ data (bin format) and obtain the
>intensity image.
>Selection of a pixel displays the decay curve but not good fit is obtained.
>
>Can someone maybe post guidelines which settings should be used?
>There is FLIMfit documentation but that does not give a protocol of the steps
>to take.
>
>All info is welcome.
>
>Best
>
>JW
>
>
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