[ome-users] [FLIMfit-users] FLIMfit is not fitting B-H data nor PQ data
Munro, Ian
i.munro at imperial.ac.uk
Fri Sep 19 10:18:44 BST 2014
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-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: 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
>>
>>
>> _______________________________________________
>> 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
>
More information about the ome-users
mailing list