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

Fri Sep 19 10:38:45 BST 2014

Hi Ian,

Thanks very much for the feedback. I might go to 3x3 smoothing; since I tend to use Nyquist sampling, my pixels are quite small anyway.

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)
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: Munro, Ian [mailto:i.munro at imperial.ac.uk]
>Sent: Friday, September 19, 2014 10:19 AM
>To: Paul Thomas (SCI)
>Cc: Borst, Jan Willem; ome-users at lists.openmicroscopy.org.uk
>Subject: Re: [ome-users] [FLIMfit-users] FLIMfit is not fitting B-H data nor PQ
>data
>
>Hi Paul
>
>It's a bit hard for me to say what's appropriate for your experiment but that
>sounds good to me.
>I would suggest, for TCSPC data & pixel by pixel fitting , changing the algorithm
>to 'Maximum likelihood' using the advanced tab.
>It does slow the fit but handles Poisson noise much better.
>It might also be worth thinking about whether you can make use of a Global
>fitting approach as you are currently smoothing the images quite heavily with
>a corresponding loss of spatial resolution.
>
>All the best
>
>Ian
>
>
>On 19 Sep 2014, at 08:34, Paul Thomas (SCI) <P.Thomas at uea.ac.uk> wrote:
>
>> 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-we
>> llcome-lab Personal web-page:
>> https://www.uea.ac.uk/biological-sciences/research/facilities/henry-we
>> llcome-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: 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
>>> @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.openmicrosco
>>> p 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
>>> @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
>>> @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
>>>
>>>
>>> _______________________________________________
>>> FLIMfit-users mailing list
>>> FLIMfit-users at lists.openmicroscopy.org.uk<mailto:FLIMfit-
>>> users at lists.openmicroscopy.org.uk><mailto:FLIMfit-
>>> users at lists.openmicroscopy.org.uk>
>>> http://lists.openmicroscopy.org.uk/mailman/listinfo/flimfit-users
>>>
>>>
>>>
>>>
>>>
>>> _______________________________________________
>>> ome-users mailing list
>>> ome-users at lists.openmicroscopy.org.uk<mailto:ome-
>>> users at lists.openmicroscopy.org.uk><mailto:ome-
>>> users at lists.openmicroscopy.org.uk>
>>> http://lists.openmicroscopy.org.uk/mailman/listinfo/ome-users
>>