Biomod/2011/TUM/TNT/Methods/FRET: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
Line 28: Line 28:


<h2>Bulk measurements</h2>
<h2>Bulk measurements</h2>
Some test bulk measurements were performed both with a photospectrometer and with a RT-PCR. Excitation and emission spectra at the photospectrometer were used to determine optimal wavelengths for FRET measurements. At the more sensitive RT-PCR, Atto 550 was excited at 545 nm and fluorescence detected at 568 nm. Atto 647N was excited at 635 nm and detected 665 nm, thus matching the conditions of the RT PCR's optical filters.  
Some test bulk measurements were performed both with a photospectrometer and with a RT-PCR. Therefore we used the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#MH_255_and_MH_256 MH_255/256 18bp double helix] with FRET labels at both ends. Thus the distance of the labels should match the Förster distance. Excitation and emission spectra at the photospectrometer were used to determine optimal wavelengths for FRET measurements. At the more sensitive RT-PCR, Atto 550 was excited at 545 nm and fluorescence detected at 568 nm. Atto 647N was excited at 635 nm and detected 665 nm, thus matching the conditions of the RT PCR's optical filters.  


<h2>Single molecule measurements</h2>
<h2>Single molecule measurements</h2>

Revision as of 16:01, 31 October 2011

<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.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;}


div.thumb { border-bottom-color: rgb(255, 255, 255); border-bottom-style: solid; border-left-color: rgb(255, 255, 255); border-left-style: solid; border-right-color: rgb(255, 255, 255); border-right-style: solid; border-top-color: rgb(255, 255, 255); border-top-style: solid; margin-bottom: 0.5em; width: auto; 

}

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

}

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"> var fullDates = new Array('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','08/31/2011','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');

$(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>


<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.3/jquery.min.js"></script>


<meta http-equiv="Content-Type" content="text/html; charset=utf-8" /> 

 <script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4.2/jquery.min.js"></script>
 <script type="text/javascript">
  $(document).ready(function(){
   $('#lside1').mouseover(function() {
    $('#lside1').stop().animate({backgroundPosition: '0px 0px'}, 1000);
   });
   $('#lside1').mouseleave(function() {
    $('#lside1').stop().animate({backgroundPosition: '-250px 0px'}, 1000);
   });
   $('#lside2').mouseover(function() {
    $('#lside2').stop().animate({backgroundPosition: '0px 0px'}, 1000);
   });
   $('#lside2').mouseleave(function() {
    $('#lside2').stop().animate({backgroundPosition: '-250px 0px'}, 1000);
   });
   $('#lside3').mouseover(function() {
    $('#lside3').stop().animate({backgroundPosition: '0px 0px'}, 1000);
   });
   $('#lside3').mouseleave(function() {
    $('#lside3').stop().animate({backgroundPosition: '-250px 0px'}, 1000);
   });
  });
 </script>

<style type="text/css"> 

  #side1 {
   width: 250px;
   height: 100px;
   position: relative;
   top: 0px;
   left: 20px;}

  #lside1 {
   width: 250px;
   height: 100px;
   background: url(http://www.tftshop.net/Testbild_100ppi.jpg) -250px 0px no-repeat;
   position: absolute;
   top: 0px;
   left: 0px;}

  #side2 {
   width: 250px;
   height: 100px;
   position: relative;
   top: 0px;
   left: 20px;}

  #lside2 {
   width: 250px;
   height: 100px;
   background: url(http://www.tftshop.net/Testbild_100ppi.jpg) -250px 0px no-repeat;
   position: absolute;
   top: 0px;
   left: 0px;}

  #side3 {
   width: 250px;
   height: 100px;
   position: relative;
   top: 0px;
   left: 20px;}

  #lside3 {
   width: 250px;
   height: 100px;
   background: url(http://www.tftshop.net/Testbild_100ppi.jpg) -250px 0px no-repeat;
   position: absolute;
   top: 0px;
   left: 0px;}

 </style>


<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 style="background-color: #cccccc"><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods"><b>Methods</b></a>
       <span>
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Folding">Folding</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Purification">Purification</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Labeling">Labeling</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#TEM">TEM</a>|
           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Fluorescence_Microscopy">Fluorescence Microscopy</a>
       </span>
 </li>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Team">Team</a></li>

<li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA">Labbook</a><!--Subnav Starts Here--> 


   <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>

</body> </html>

FRET

Förster resonance energy transfer (FRET) describes the energy transfer through nonradiative dipole–dipole interaction between two chromophores. If the emission spectrum of one chromophore overlaps with the excitation spectrum of the other chromophore and both chromophores are in proximity (aprx. 10nm) to each other, an excited donor chromophore can transfer energy to an acceptor chromophore resulting in fluorescence with the emission wavelength of the acceptor chromophore.

The FRET efficiency ([math]\displaystyle{ E }[/math]) is the fraction of energy transfer occurring per donor excitation event:

[math]\displaystyle{ E = \frac{k_{ET}}{k_f+k_{ET}+\sum{k_i}} }[/math]

where [math]\displaystyle{ k_{ET} }[/math] is the rate of energy transfer, [math]\displaystyle{ k_{f} }[/math] the radiative decay rate and the [math]\displaystyle{ k_{i} }[/math] are the rate constants of any other de-excitation pathway.

The FRET efficiency depends on many parameters that can be grouped as follows:

  • The distance and orientation between the donor and the acceptor
  • The spectral overlap of the donor emission spectrum and the acceptor absorption spectrum.
  • The relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment.

[math]\displaystyle{ E }[/math] depends on the donor-to-acceptor separation distance [math]\displaystyle{ r }[/math] with an inverse 6th power law due to the dipole-dipole coupling mechanism:

[math]\displaystyle{ E=\frac{1}{1+(r/R_0)^6}\! }[/math]

with [math]\displaystyle{ R_0 }[/math] being the Förster distance of this pair of donor and acceptor, i.e. the distance at which the energy transfer efficiency is 50%.

The FRET efficiency relates to the fluorescence lifetime of the donor molecule as follows:

[math]\displaystyle{ E = 1 - {\tau'_{\rm D}}/{\tau_{\rm D}} \! }[/math]

where [math]\displaystyle{ \tau'_{\rm D} }[/math] and [math]\displaystyle{ \tau_{\rm D} }[/math] are the donor fluorescence lifetimes in the presence and absence of an acceptor, respectively, or as

[math]\displaystyle{ E = 1 - {F\,'_{\rm D}}/{F_{\rm D}} \! }[/math]

where [math]\displaystyle{ F\,'_{\rm D} }[/math] and [math]\displaystyle{ F_{\rm D} }[/math] are the donor fluorescence intensities with and without an acceptor, respectively.

Bulk measurements

Some test bulk measurements were performed both with a photospectrometer and with a RT-PCR. Therefore we used the MH_255/256 18bp double helix with FRET labels at both ends. Thus the distance of the labels should match the Förster distance. Excitation and emission spectra at the photospectrometer were used to determine optimal wavelengths for FRET measurements. At the more sensitive RT-PCR, Atto 550 was excited at 545 nm and fluorescence detected at 568 nm. Atto 647N was excited at 635 nm and detected 665 nm, thus matching the conditions of the RT PCR's optical filters.

Single molecule measurements

Immobilization

For single molecule fluorescence microscopy, the origamis need to be immobilized. We need similar orientations on the surface. The structures should stand on the base of the "U", since flat lying origamis might display limited flexibility. For this aim, seven additional adapter staples were added at the bottom of the structures. 21 bp of these staples reach beyond the base. When preparing samples for the fluorescence microscope, the slide is incubated with neutravidin and then with biotinylated staples. These are complementary to the free base pairs of the adapters, so hybridization can take place and the origamis are orientated in the wanted direction.

Fluorescence microscope setup

  • Green laser: Excitation at 532 nm
  • Red laser: Excitation at 640 nm


filter, wavelengths, etc.

Evaluation of data

http://openwetware.org/index.php?title=Biomod/2011/TUM/TNT/Methods/FRET&action=edit


Inhalt:

material and methods
suppliers


FRET erklären. Links zu Results und Labbook.


Retrieved from "https://openwetware.org/mediawiki/index.php?title=Biomod/2011/TUM/TNT/Methods/FRET&oldid=557377"