Biomod/2011/TUM/TNT/LabbookA/evaluation of TIRF data in Matlab

From OpenWetWare
Jump to: navigation, search

<html>

  <style>
  1. column-one { display:none; width:0px;}

.container{background-color: #f5f5f5; margin-top:50px} .OWWNBcpCurrentDateFilled {display: none;}

  1. content { width: 0px; margin: 0 auto auto 0; padding: 1em 1em 1em 1em; align: center;}
  1. column-content {width: 0px; float: left; margin: 0 0 0 0;padding: 0;}

.firstHeading {display:none; width:0px;}

  1. globalWrapper{ width:1280px;}

div.tright { border-bottom-width: 0em; border-left-width: 0px; border-right-width: 0px; border-top-width: 0em; clear: right; float: right;

}

div.tleft { border-bottom-width: 0em; border-left-width: 0px; border-right-width: em; border-top-width: 0em; clear: left; float: left; margin-right: 0.5em;

}

div.tnone { border-bottom-width: 0em; border-left-width: 0px; border-right-width: em; border-top-width: 0em; }

  1. column-one {display:none; width:0px;background-color: #ffffff;}
  1. content{ margin: 0 0 0 0; padding: 1em 1em 1em 1em; position: center; width: 800px;background-color: #f5f5f5; }

.container{ width: 800px; margin: auto; background-color: #ffffff; text-align:justify; font-family: helvetica, arial, sans-serif; color:#555555; margin-top:25px; }

  1. bodyContent{ width: 1267px; align: center; background-color: #ffffff;}
  1. column-content{width: 1280px;background-color: #f5f5f5;}

.firstHeading { display:none;width:0px;background-color: #ffffff;}

  1. header{position: center; width: 800px;background-color: #ffffff;}
  1. footer{position: center;}

<style type="text/css"> body {

   font-family: helvetica, arial, sans-serif;
   color: black;

background:#f5f5f5;

 }
ul#topnav {

margin: 0 0 0 0px ; padding: 0 0 0 0px; float: left; width: 800px; list-style: none; position: relative;

       font-family: helvetica, arial, sans-serif;

font-size: 12px; background: #f5f5f5;

       height: 5px;

} ul#topnav li { float: left; margin: 0; padding: 0; border-right: 1px solid #ffffff; /*--Divider for each parent level links--*/ } ul#topnav li a { padding: 11px 15px; display: block; color: #333333; text-decoration: none; } ul#topnav li:hover { background: #f5f5f5;} /*--Notice the hover color is on the list item itself, not on the link. This is so it can stay highlighted even when hovering over the subnav--*/

ul#topnav li span { float: left; padding: 15px 0; position: absolute; left: 0; top:30px; display: inline; /*--Hide by default--*/ width: 800px; background: #cccccc; color: #333333; /*--Bottom right rounded corner--*/ -moz-border-radius-bottomright: 5px; -khtml-border-radius-bottomright: 5px; -webkit-border-bottom-right-radius: 5px; /*--Bottom left rounded corner--*/ -moz-border-radius-bottomleft: 5px; -khtml-border-radius-bottomleft: 5px; -webkit-border-bottom-left-radius: 5px; } ul#topnav li:hover span { display: block; } /*--Show subnav on hover--*/ ul#topnav li span a { display: inline;} /*--Since we declared a link style on the parent list link, we will correct it back to its original state--*/ ul#topnav li span a:hover {text-decoration: underline;}


  1. abstractandlinks{

display:block; width:800px; background-color:#f5f5f5; text-align:justify;font-family: helvetica, arial, sans-serif; color:#555555; margin-top:0px; }

  1. innerBox

{ display: block; padding: 5px; }

  1. abstract{

font-family: helvetica, arial, sans-serif; width: 490px; margin-top: 20px; padding:0px 5px; float: left;background-color:#f5f5f5; ; }

  1. links{

font-family: helvetica, arial, sans-serif; width: 290px; margin-top: 20px; padding:0px 5px; float: left; background-color:#f5f5f5;; } .clear { clear:both; }

  1. tour{

font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; margin-top: 1em, margin-bottom: 1em; float: left;background:#f5f5f5; font-size: 2em; }

  1. struktur{

font-family: helvetica, arial, sans-serif; width: 790px; padding:0px 5px; float: left;background:#f5f5f5; }

  1. toctitle {align: left; text-align: left; }
  2. table.toc {background-color:"#5f5f5f";align: left; text-align: left;}
  3. toc #toctitle, .toc #toctitle, #toc .toctitle, .toc .toctitle {

text-align: left;

}
  1. toc, .toc, .mw-warning {

background-color: #f5f5f5; border-bottom-color: rgb(170, 170, 170); border-bottom-style: solid; border-bottom-width: 0px; border-left-color: rgb(170, 170, 170); border-left-style: solid; border-left-width: 0px; border-right-color: rgb(170, 170, 170); border-right-style: solid; border-right-width: 0px; border-top-color: rgb(170, 170, 170); border-top-style: solid; border-top-width: 0px; font-size: 105%; padding-bottom: 5px; padding-left: 5px; padding-right: 5px; padding-top: 5px; align: left; text-align: left; display: left

}


</style>

<head>

<script type="text/javascript">


$(document).ready(function() {

$("ul#topnav li").hover(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav

} ,

$("ul#topnav li").oneclick(function() { //Hover over event on list item $(this).css({ 'background' : '#cccccc'; 'display' : 'inline'}); //Add background color and image on hovered list item $(this).find("span").show(); //Show the subnav

} ,

function() { //on hover out...


$(this).css({ 'background' : 'none'}); //Ditch the background

//$(this).find("span").hide(); //Hide the subnav });

}); </script>


<title>

       TUM NanU - Home   

</title> </head>

<body background-color="#F0F0F0"> <!-- Header--> <div class="container">

   <div id="header"> <img src="http://openwetware.org/images/5/5c/Header2.png" /> </div> 
   

<!--Menue--> <ul id="topnav">

   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Home">Home</a></li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Project">Project</a>        
      </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Results">Results & Discussion</a>        
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods">Methods</a></li>
      
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Team">Team</a></li>
<li style="background-color: #cccccc"><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA"><b>Labbook</b></a><!--Subnav Starts Here-->
       <span>
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA">Main Page</a>||
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page">Structure</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/list_TEM_pictures">TEM</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/TIRF">Fluorescence Microscopy</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Glossary">Glossary</a>
        </span>
   </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras">Extras</a>
       
  </li>

</ul> <p> <br> </p> <p> <br> </p> <p> <br> </p> </html>

<html>

<html> <head> <script type= "text/javascript">/*<![CDATA[*/ var skin = "monobook"; var stylepath = "/skins"; var wgArticlePath = "/wiki/$1"; var wgScriptPath = ""; var wgScript = "/index.php"; var wgVariantArticlePath = false; var wgActionPaths = []; var wgServer = "http://openwetware.org"; var wgCanonicalNamespace = ""; var wgCanonicalSpecialPageName = false; var wgNamespaceNumber = 0; var wgPageName = "Biomod/2011/TUM/TNT/LabbookA/2011"; var wgTitle = "Biomod/2011/TUM/TNT/LabbookA/2011 var wgAction = "view"; var wgArticleId = "103708"; var wgIsArticle = true; var wgUserName = "Alexander Hipp"; var wgUserGroups = ["*", "user", "autoconfirmed"]; var wgUserLanguage = "en"; var wgContentLanguage = "en"; var wgBreakFrames = false; var wgCurRevisionId = "543703"; var wgVersion = "1.13.2"; var wgEnableAPI = true; var wgEnableWriteAPI = false; var wgMWSuggestTemplate = "http://openwetware.org/api.php?action=opensearch\x26search={searchTerms}\x26namespace={namespaces}"; var wgDBname = "owwdb"; var wgSearchNamespaces = [0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 100, 101, 110, 111]; var wgMWSuggestMessages = ["with suggestions", "no suggestions"]; var wgRestrictionEdit = []; var wgRestrictionMove = []; var wgAjaxWatch = {"watchMsg": "Watch", "unwatchMsg": "Unwatch", "watchingMsg": "Watching...", "unwatchingMsg": "Unwatching..."}; /*]]>*/</script>

<script type="text/javascript" src="/skins/common/wikibits.js?164"><!-- wikibits js --></script> <!-- Head Scripts --> <script type="text/javascript" src="/js/calendar.js"></script> <script type="text/javascript"> function lnCalendar(){

 var calm1 = new CalendarPopup('m1');
 var wikiPage = 'Biomod/2011/TUM/TNT/LabbookA';
 var redirPage = '/wiki/Special:Redir/'+wikiPage;
 var fullDates = new Array('08/11/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','09/01/2011','09/02/2011','09/06/2011','09/12/2011','09/13/2011','09/14/2011','09/15/2011','09/16/2011','09/19/2011','09/20/2011','09/21/2011','09/27/2011','09/28/2011','09/29/2011','09/30/2011','10/05/2011','10/06/2011','10/07/2011','10/10/2011','10/12/2011','10/13/2011','10/14/2011','10/17/2011','10/18/2011','10/19/2011','10/20/2011','10/21/2011','10/26/2011','10/27/2011','10/28/2011','10/30/2011','10/31/2011');
 calm1.setDateFormat('yyyy/MM/dd');
 calm1.setCssPrefix('OWWNB');
 calm1.setSingleMonth('8');
 calm1.setYear('2011');
 calm1.setReadOnly('N');
 calm1.setTodayText('');
 calm1.setUrlPrefix(redirPage);
 for(i = 0; i < fullDates.length; i++)calm1.addFilledDates(fullDates[i]);
 calm1.showCalendar('m1');
 var calm2 = new CalendarPopup('m2');

('08/11/2011','08/12/2011','08/16/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','09/01/2011','09/02/2011','09/06/2011','09/12/2011','09/13/2011','09/14/2011','09/15/2011','09/16/2011','09/19/2011','09/20/2011','09/21/2011','09/27/2011','09/28/2011','09/29/2011','09/30/2011','10/05/2011','10/06/2011','10/07/2011','10/10/2011','10/12/2011','10/13/2011','10/14/2011','10/17/2011','10/18/2011','10/19/2011','10/20/2011','10/21/2011','10/26/2011','10/27/2011','10/28/2011','10/30/2011','10/31/2011');

 calm2.setDateFormat('yyyy/MM/dd');
 calm2.setCssPrefix('OWWNB');
 calm2.setSingleMonth('9');
 calm2.setYear('2011');
 calm2.setReadOnly('N');
 calm2.setTodayText('');
 calm2.setUrlPrefix(redirPage);
 for(i = 0; i < fullDates.length; i++)calm2.addFilledDates(fullDates[i]);
 calm2.showCalendar('m2');
 var calm3 = new CalendarPopup('m3');

('08/11/2011','08/12/2011','08/16/2011','08/17/2011','08/23/2011','08/24/2011','08/25/2011','08/29/2011','08/30/2011','09/01/2011','09/02/2011','09/06/2011','09/12/2011','09/13/2011','09/14/2011','09/15/2011','09/16/2011','09/19/2011','09/20/2011','09/21/2011','09/27/2011','09/28/2011','09/29/2011','09/30/2011','10/05/2011','10/06/2011','10/07/2011','10/10/2011','10/12/2011','10/13/2011','10/14/2011','10/17/2011','10/18/2011','10/19/2011','10/20/2011','10/21/2011','10/26/2011','10/27/2011','10/28/2011','10/30/2011','10/31/2011');

 calm3.setDateFormat('yyyy/MM/dd');
 calm3.setCssPrefix('OWWNB');
 calm3.setSingleMonth('10');
 calm3.setYear('2011');
 calm3.setReadOnly('N');
 calm3.setTodayText('');
 calm3.setUrlPrefix(redirPage);
 for(i = 0; i < fullDates.length; i++)calm3.addFilledDates(fullDates[i]);
 calm3.showCalendar('m3');

} </script> <script type="text/javascript">addOnloadHook( lnCalendar )</script> <script type="text/javascript">function CalendarPageConfirmCreate(date, url){var answer = confirm("Create entry for "+date+"?");if (answer){window.location = url;}}</script> <script type="text/javascript" src="/skins/common/ajax.js?164"></script> <script type="text/javascript" src="/ext/AjaxShowEditors/AjaxShowEditors.js"></script> <script type="text/javascript" src="/skins/common/ajaxwatch.js?164"></script> <script type="text/javascript" src="/skins/common/mwsuggest.js?164"></script> <script type="text/javascript" src="/index.php?title=-&amp;action=raw&amp;smaxage=0&amp;gen=js&amp;useskin=monobook"><!-- site js --></script>


<style type="text/css">/*<![CDATA[*/ @import "/index.php?title=MediaWiki:Common.css&amp;usemsgcache=yes&amp;action=raw&amp;ctype=text/css&amp;smaxage=18000"; @import "/index.php?title=MediaWiki:Monobook.css&amp;usemsgcache=yes&amp;action=raw&amp;ctype=text/css&amp;smaxage=18000"; @import "/index.php?title=-&amp;action=raw&amp;gen=css&amp;maxage=18000&amp;smaxage=0&amp;ts=20111012100110&amp;useskin=monobook"; @import "/index.php?title=User:Alexander_Hipp/monobook.css&amp;action=raw&amp;ctype=text/css"; /*]]>*/</style> <script type="text/javascript" src="/index.php?title=User:Alexander_Hipp/monobook.js&amp;action=raw&amp;ctype=text/javascript"></script> </head>

</html>

General Outline

In order to analyze the acquired FRET movies and extract accurate FRET efficiencies, a homemade Matlab software was used.

The software automatically picks spots, based on user chosen thresholds and tracks them throughout the whole movie. It integrates a circular region of 5 pixel per spot in each frame and generates intensity traces. A mapping between the donor and acceptor detection channel was done to colocalize corresponding tracks.

Traces showing a donor as well as an acceptor signal were automatically extracted and afterwards manually selected for typical bleaching characteristics; I.e. bleaching of the acceptor is anticorrelated to increase in donor intensity and bleaching of the donor at last. This is necessary to determine background intensities, calculate the gamma factor and therefore obtain accurate FRET efficiencies. Hereby, intensity levels were extracted by fitting (multi-)step funtions.

Calculating accurate FRET

Background corrected intensity levels for the donor Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle I_D} and acceptor Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle I_{D \rightarrow A}} when both dyes were still present were obtained as described above. The true energy transfer is then given by:

Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle E_t = \frac{I_{D \rightarrow A}}{I_{D \rightarrow A}+ \gamma \; I_D}}

where Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \gamma} is used to correct for differences in efficiencies between donor and acceptor channel as well as for varying quantum yield. It is calculated from the change in donor intensity Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \Delta I_D} and acceptor intensity Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \Delta I_{D\rightarrow A}} as follows:

Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle \gamma = \frac{\Delta I_{D \rightarrow A}}{\Delta I_D}}