<html>
<head>
<meta http-equiv="Content-Type" content="text/html; charset=Windows-1252">
</head>
<body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;">
Hi Ewan
<div><br>
</div>
<div>For the moment I’m just forwarding this to the users list in case somebody can answer immediately.</div>
<div>Otherwise I’ll have a look as soon as I can.</div>
<div><br>
</div>
<div>All the best </div>
<div><br>
</div>
<div>Ian<br>
<div><br>
<div>Begin forwarded message:</div>
<br>
<blockquote type="cite">
<div>
<div style="font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; font-size: 14px; font-family: Calibri, sans-serif;">
<div>Hi </div>
<div><br>
</div>
<div>I’ve just been doing some double exponential fitting with FLIMFit and ran into a couple of “queries”…</div>
<div><br>
</div>
<div>1. When I used a pixel-by-pixel method you’d get a longer than expected lifetime and and shorter than expected lifetime (e.g. For a gfp-Tag-RFP fret pair, should be about 2.1 ns gfp lifetime, I got t1 = 3.2 ns and t2 = ~600 ps with b1 = ~0.3 and b2 = ~0.7),
however, the weighted mean lifetime is the correct value?</div>
<div><br>
</div>
<div>2. Sometime the beta were negative for b2 and over 1 for b1 e.g. b1 = 1.41 and b2 = -0.42?</div>
<div><br>
</div>
<div>I’m guessing that this is because the model is finding it hard to fit perhaps through lack of signal?</div>
<div><br>
</div>
<div>When I fit the same data using global fitting (but locally vary the contributions) it behaves a bit better.</div>
<div><br>
</div>
<div>
<div style="margin: 0px; font-size: 12px; font-family: 'Lucida Grande';">The smodel settings I used were: Global fitting, Fit Contributions = Fitted locally, Fit Reference = Fitted, Fit IRF Shift = Fitted. Stray light tab: Offest = Fitted gloabally, Scatter
= Fitted gloabally, TV Background = Fitted globally.</div>
<div style="margin: 0px; font-size: 12px; font-family: 'Lucida Grande';"><br>
</div>
<div style="margin: 0px; font-size: 12px; font-family: 'Lucida Grande';">The fit results are:</div>
<div style="margin: 0px; font-size: 12px; font-family: 'Lucida Grande';"><br>
</div>
<p style="margin: 0px; font-size: 12px; font-family: 'Lucida Grande';"></p>
<table border="0" cellpadding="0" cellspacing="0" width="421" style="border-collapse: collapse; width: 421pt;">
<colgroup><col width="53" span="3" style="width: 53pt;"><col width="80" style="width: 80pt;"><col width="75" style="width: 75pt;"><col width="107" style="width: 107pt;"></colgroup>
<tbody>
<tr height="14" style="height: 14pt;">
<td height="14" width="53" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; height: 14pt; width: 53pt;">
</td>
<td class="xl63" width="53" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; width: 53pt;">
tau 1</td>
<td class="xl63" width="53" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; width: 53pt;">
tau 2</td>
<td class="xl63" width="80" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; width: 80pt;">
b1</td>
<td class="xl63" width="75" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; width: 75pt;">
b2</td>
<td class="xl63" width="107" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; width: 107pt;">
weighted mean tau</td>
</tr>
<tr height="14" style="height: 14pt;">
<td height="14" class="xl63" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; height: 14pt;">
mTagRFP</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
2374.9365</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
101.5567</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
1.04607</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
-0.0460605</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
2478.4242</td>
</tr>
<tr height="14" style="height: 14pt;">
<td height="14" class="xl63" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; height: 14pt;">
control</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
2203.113</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
326.7455</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
0.60874</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
0.39126</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
2034.0543</td>
</tr>
<tr height="14" style="height: 14pt;">
<td height="14" class="xl63" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; height: 14pt;">
<br>
</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
<br>
</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
<br>
</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
<br>
</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
<br>
</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
<br>
</td>
</tr>
<tr height="14" style="height: 14pt;">
<td height="14" class="xl63" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 700; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap; height: 14pt;">
wt - 2</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
2222.8235</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
74.7949</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
1.5714</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
-0.571393333</td>
<td align="right" style="padding-top: 1px; padding-right: 1px; padding-left: 1px; font-size: 11pt; font-weight: 400; font-style: normal; text-decoration: none; font-family: Calibri, sans-serif; vertical-align: bottom; border: none; white-space: nowrap;">
2258.200767</td>
</tr>
</tbody>
</table>
</div>
<div><br>
</div>
<div> </div>
<div> Where mTagRFP, is just GFP only so 2.5’ish ns expected, control is positive FRET so about 2 ns and wt-2 is the wild-type free to vary FRET sample (so somewhere between mTagRFP and control sample lifetimes).</div>
<div><br>
</div>
<div>Does this behaviour make sense to you?</div>
<div><br>
</div>
<div>Any help or insight would be really useful :-) </div>
<div><br>
</div>
<div>Hows things going otherwise? I shall email you the journal DOI when the paper is published that this is going into.</div>
<div><br>
</div>
<div>Speak to you soon,</div>
<div><br>
</div>
<div>Ewan</div>
</div>
</div>
</blockquote>
</div>
<br>
</div>
</body>
</html>