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<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">HI Mayur,<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">For a double exponential analysis I would advise using a measured IRF or a reference dye measurement.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">A double exponential analysis is much more dependent on an accurate IRF measurement than a single exponential analysis.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Could you let me know if you are using a PMT or HyD for the FLIM measurement?<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Another factor that could lead to an erroneously high long lifetime would be background light.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">I would suggest making two background measurements:<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><span style="mso-list:Ignore">1.<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">With the laser turned off to measure the room light/detector dark counts.
<o:p></o:p></span></p>
<p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><![if !supportLists]><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><span style="mso-list:Ignore">2.<span style="font:7.0pt "Times New Roman"">
</span></span></span><![endif]><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"> A media-only measurement with no cells at the same laser power and exposure time to determine if there is background fluorescence from the media<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">If you can make these control measurements I’d be happy to walk you through troubleshooting your analysis.
<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Thanks<o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D">Sean<o:p></o:p></span></p>
<p class="MsoNormal" style="mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;line-height:14.0pt;background:white">
<b><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#333333">Dr. Sean Warren </span></b><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#333333">| Research Officer<br>
Invasion and Metastasis<br>
Cancer Division<br>
<b>Garvan Institute of Medical Research </b><br>
The Kinghorn Cancer Centre,<b> </b>370 Victoria Street, Darlinghurst, NSW 2010</span><span style="font-size:10.0pt;font-family:"Arial","sans-serif";color:#333333"><o:p></o:p></span></p>
<p class="MsoNormal"><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><o:p> </o:p></span></p>
<p class="MsoNormal"><b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif"">From:</span></b><span lang="EN-US" style="font-size:10.0pt;font-family:"Tahoma","sans-serif""> FLIMfit-users [mailto:flimfit-users-bounces@lists.openmicroscopy.org.uk]
<b>On Behalf Of </b>Mayur Vadhvani<br>
<b>Sent:</b> Wednesday, 22 March 2017 5:36 AM<br>
<b>To:</b> flimfit-users@lists.openmicroscopy.org.uk<br>
<b>Cc:</b> markosostar@gmail.com<br>
<b>Subject:</b> [FLIMfit-users] FLIM-FRET image analysis<o:p></o:p></span></p>
<p class="MsoNormal"><o:p> </o:p></p>
<div>
<p class="MsoNormal">Dear All,<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
</div>
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<p class="MsoNormal">I am a neurobiologist/cell biologist at Institute of Biochemistry, Charite Medical University, Berlin.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal">I am trying to study the interaction between 2 proteins (donor GFP-tagged protein present in nucleus and cytoplasm whereas acceptor mCherry-tagged protein present only in the nucleus) using FLIM-FRET and am naive to the detailed analysis
of my samples using FLIMfit.<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
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<p class="MsoNormal">We are using the Leica SP5 confocal system with Picoharp300 (Picoquant) at 80 MHz for acquiring our data and using FLIMfit to analyze our images.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
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<div>
<p class="MsoNormal">I am going through the following steps to work on the data (sample image):<o:p></o:p></p>
</div>
<div>
<p class="MsoNormal">1. I assess the decay at a given pixel and import pre-defined IRF from SymphoTime64.<o:p></o:p></p>
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<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal"><img width="544" height="340" id="_x0000_i1025" src="cid:image001.png@01D2A70F.B0DE1C60" alt="Inline image 2"><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
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<div>
<p class="MsoNormal">2. As the pixel number is low, I use 5x5 or 7x7 smoothing (7x7 in this example). <span style="font-size:9.5pt"> After that I select segments in the cell (nucleus and cytoplasm respectively) and perform a mono-exponential fitting, which
gives me this:</span><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><img width="544" height="340" id="_x0000_i1026" src="cid:image002.png@01D2A70F.B0DE1C60" alt="Inline image 3"></span><o:p></o:p></p>
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<div>
<p class="MsoNormal"><o:p> </o:p></p>
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<p class="MsoNormal"><span style="font-size:9.5pt">3. Since I have FRET data, I perform a bi-exponential fit and use the Global fitting approach which gives me this:</span><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><img width="544" height="340" id="_x0000_i1027" src="cid:image003.png@01D2A70F.B0DE1C60" alt="Inline image 4"></span><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">4. When I look at the parameters of selected segments, I get the following info:</span><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><img width="544" height="340" id="_x0000_i1028" src="cid:image004.png@01D2A70F.B0DE1C60" alt="Inline image 6"></span><o:p></o:p></p>
</div>
<div>
<p class="MsoNormal"><o:p> </o:p></p>
</div>
<div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">1- Cell 1/cytoplasm<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">2- Cell 1/nucleus<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">3- Cell 2/nucleus<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">4- Cell 2/cytoplasm<o:p></o:p></span></p>
</div>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
<div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">I had the following questions concerning my analysis:<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">1. I wanted to confirm whether the tau_2 values I get are the donor lifetime values for the given segments (2.35 ns in cytoplasm vs 2.22/2.20 ns in nucleus)? Because I expect FRET only in the nucleus, I would
see a decrease in the lifetime values only for the nucleus as compared to the cytoplasm from the same cell (which would serve as my internal control).<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">2. I don't quite understand the high tau_1 values that I observe. What could be the explanation for that?<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">3.Can I rely on the values presented above? I am concerned about the IRF as it is predetermined value that I am using. As suggested by Ewan McGhee in response to Marko Šoštar's query, I can try to use FITC
to measure IRF of our setup.<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">4. Is there anything I can make my data acquisition or analysis more robust?<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">I would really appreciate any feedback on this.<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Thank you very much.<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Regards,<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Mayur Vadhvani<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Postdoctoral Researcher<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Institute of Biochemistry<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Charite Medical University<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Berlin<o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Germany<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.5pt">PS: <o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">Dear Marko, <o:p></o:p></span></p>
</div>
<div>
<p class="MsoNormal"><span style="font-size:9.5pt">I am also including you in the email as I am also using the Leica/PicoQuant system for my image acquisition.<o:p></o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
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<p class="MsoNormal"><span style="font-size:9.5pt"><o:p> </o:p></span></p>
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