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<div>Hi Markus</div>
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<div>Firstly I hope it’s ok if I cc this to the FLIMfit-users mailing list </div>
<div><a href="http://lists.openmicroscopy.org.uk/mailman/listinfo/flimfit-users">lists.openmicroscopy.org.uk/mailman/listinfo/flimfit-users</a></div>
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<div>just in case anyone might have similar questions.</div>
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<div>An IRF gives the software 2 important pieces of information. </div>
<div>It allows it to estimate the ‘distortion’ introduced by the instrument and ,crucially, to estimate the position, in time , of the excitation pulse.</div>
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<div>In the absence of an irf, FLIMfit assumes a “delta function-irf” which i.e. a perfect detector.</div>
<div>The Excitation pulse is then assumed to be at the first non-zero point in your data (after all, given a perfect detector, no fluorescence can be detected before the excitation pulse).</div>
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<div>In the absence of a measured irf you can use this to do a “tail-fit” .</div>
<div>To do this, estimate the peak of the decay by eye & exclude all earlier points from the decay using the time-min box on the data tab.</div>
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<div>the attached screen shots illustrate this.</div>
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<div>Beware though. A tail-fit is fine for qualitative analysis. ( does area a) have a shorter lifetime than area b) ).</div>
<div>For quantitative analysis it is very dangerous as you are entering a crucial parameter ( the excitation pulse position ) by ‘eye’.</div>
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<div>To select a region use the shape tools (square polygon & ellipse shapes) at top left.</div>
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<div>Regards</div>
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<div>Ian</div>
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<div><img height="514" width="774" apple-width="yes" apple-height="yes" apple-inline="yes" id="E304D1E9-CB83-4568-8C90-2882BB5645DB" src="cid:CEFAA302-69A9-457D-B02E-A99762A13589"></div>
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<div>Exclude data before peak.</div>
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<div><img height="520" width="774" apple-width="yes" apple-height="yes" apple-inline="yes" id="F151B5A4-85E4-4E72-8AF2-E3BB11C5B181" src="cid:FB65B70A-8BFF-4FEC-BE10-56A79F3EBC81"></div>
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<div>Fit ! Note red line & residuals.</div>
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<div><img height="516" width="774" apple-width="yes" apple-height="yes" apple-inline="yes" id="C662CE9C-1394-4088-95C0-AAC2432F84FB" src="cid:178D0F88-6EB9-4809-B03B-3F09A229BFB2"></div>
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<div>On 12 Sep 2014, at 14:53, Markus Posch <<a href="mailto:m.posch@dundee.ac.uk">m.posch@dundee.ac.uk</a>> wrote:</div>
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<blockquote type="cite">Hi Ian-<br>
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thanks for your help in the first place!<br>
I managed to get hold of the correct TCSPC data - but still much lost in the data analysis.<br>
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Particularly I am not sure if I am missing out an IRF (instrument response function).<br>
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We think want to measure the half-lives with small areas - so basically within an ROI. Is it possible to define an ROI and get the T-half within this ROI?<br>
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For the moment we having two images only (control and FRET cell) for a brief proof of concept.<br>
Eventually and for statistical reason we’d have to do may more. So potentially displaying those in a histogram of distribution of lifetimes in all ROIs measured.<br>
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I have attached brief figure how we think we wanted to represent data (numbers in table are made up).<br>
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Is such analysis possible? One could do that on the LaVison software at the Beatson, if I remember correctly.<br>
Could you please guide me through a short workflow?<br>
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Data I have are two OME-tifs (of the TSCPC recording) - IRF needed?<br>
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Many thanks,<br>
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Markus<br>
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The University of Dundee is a registered Scottish Charity, No: SC015096<br>
<span><TCSPC.pdf></span><br>
<span><FLIM-FRET_histo.pdf></span><br>
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On 12 Sep 2014, at 12:14, Munro, Ian <<a href="mailto:i.munro@imperial.ac.uk">i.munro@imperial.ac.uk</a>> wrote:<br>
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<blockquote type="cite">Hello Marcus<br>
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I was concerned that FLIMfit wasn’t able to handle your data but from what you say, perhaps the first step is to check that it actually is time-resolved. We do have some FLIMfit users at Beatson already so perhaps they may be able to help.<br>
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Unfortunatley FLIMfit is principally a time-domain tool in terms of fitting.<br>
It might, however, be of use in terms of visual examination of the data.<br>
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Colleagues have looked at converting data between time-domain & frequency-domain (FFT I think) in order to compare the two approaches but I haven’t been at all involved.
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I could put you in touch if that would help.<br>
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All the best<br>
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Ian<br>
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On 12 Sep 2014, at 10:38, Markus Posch <<a href="mailto:m.posch@dundee.ac.uk">m.posch@dundee.ac.uk</a>> wrote:<br>
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<blockquote type="cite">Hello Ian-<br>
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thanks for your mail and may thanks for offering your help!<br>
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We did some preliminary FLIM experiments at the Beatson Institute and are trying to get a feel for FLIMfit on these data. Early days, so we still have a steep learning curve and all needs to make slowly sense to us.<br>
Data is from the LaVison TCSPC and the Lambert Inst. LIFA system.<br>
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We have tried to upload some TCSPC data into FLIMfit, but it appears this was only RGB-Tif snapshots, rather than the actual dataset.<br>
Petr has identified there was clearly no time dimension in the data. - I will try to check with Beatson on the data!<br>
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I could also upload the data (this was just a pilot with a handful of cells) on OMERO. What server would make sense?<br>
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Your offer to help is certainly most welcome!<br>
Will FLIMfit work with TSCPC data only or also process frequency modulated data, as from the LIFA system?<br>
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Many thanks!<br>
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Markus<br>
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The University of Dundee is a registered Scottish Charity, No: SC015096<br>
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