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