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

Thu Sep 18 20:41:59 BST 2014

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>
I: www.biochemistry.wur.nl<http://www.biochemistry.wur.nl>, www.mscwu.wur.nl<http://www.mscwu.wur.nl>
 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>> 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>> 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>
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: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>
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>> 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>> 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>>
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>> 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>
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>
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>
http://lists.openmicroscopy.org.uk/mailman/listinfo/ome-users