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

Thu Sep 18 14:00:35 BST 2014

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
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] On Behalf Of Munro, Ian
>Sent: Thursday, September 18, 2014 1:55 PM
>To: 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 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> 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> 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>
>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> 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
>>>>>> http://lists.openmicroscopy.org.uk/mailman/listinfo/flimfit-users
>>>>>
>>>>
>>>
>>
>
>_______________________________________________
>ome-users mailing list
>ome-users at lists.openmicroscopy.org.uk
>http://lists.openmicroscopy.org.uk/mailman/listinfo/ome-users