<div dir="ltr">Dear Sean,<div><br></div><div>Thank you very much for your reply.</div><div><br></div><div>We are using the PMT for our FLIM measurement. </div><div><br></div><div>I will follow-up on the other suggestions made by you and get back to you as soon as I have the results.</div><div><br></div><div>Thank you.</div><div><br></div><div>regards,</div><div>Mayur</div><div><br></div></div><div class="gmail_extra"><br><div class="gmail_quote">On Mon, Mar 27, 2017 at 1:49 PM, Munro, Ian via FLIMfit-users <span dir="ltr"><<a href="mailto:flimfit-users@lists.openmicroscopy.org.uk" target="_blank">flimfit-users@lists.openmicroscopy.org.uk</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">



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Hi Both
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<div>If you’re both happy I feel that it would be useful if any “walkthrough” ended up accessible for others, either via e-mails on this list</div>
<div>or, if a video-chat, recorded and uploaded somewhere.</div>
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<div>Thanks</div><span class="HOEnZb"><font color="#888888">
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<div>Ian</div></font></span><span class="">
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<div>On 27 Mar 2017, at 05:34, Sean Warren via FLIMfit-users <<a href="mailto:flimfit-users@lists.openmicroscopy.org.uk" target="_blank">flimfit-users@lists.<wbr>openmicroscopy.org.uk</a>> wrote:</div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">HI Mayur,<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">For a double exponential analysis I would advise using a measured IRF or a reference dye measurement.<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">A double exponential analysis is much more dependent on an accurate IRF measurement than a single exponential analysis.<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">Could you let me know if you are using a PMT or HyD for the FLIM measurement?<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">Another factor that could lead to an erroneously high long lifetime would be background light.<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">I would suggest making two background measurements:<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><span>1.<span style="font-style:normal;font-variant-caps:normal;font-weight:normal;font-size:7pt;line-height:normal;font-family:'Times New Roman'">      <span class="m_-3144024773048725649Apple-converted-space"> </span></span></span></span><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">With
 the laser turned off to measure the room light/detector dark counts.<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><span>2.<span style="font-style:normal;font-variant-caps:normal;font-weight:normal;font-size:7pt;line-height:normal;font-family:'Times New Roman'">      <span class="m_-3144024773048725649Apple-converted-space"> </span></span></span></span><span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"> 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<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)"><u></u> <u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">If you can make these control measurements I’d be happy to walk you through troubleshooting your analysis.<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">Thanks<u></u><u></u></span></div>
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<span style="font-size:11pt;font-family:Calibri,sans-serif;color:rgb(31,73,125)">Sean<u></u><u></u></span></div>
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