Biomod/2011/TUM/TNT/Results: Difference between revisions

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Though quantitative evidence is a bit tricky because of the calibration and the fact that one pixel of the pictures equals 101.03nm  a qualitative evidence can be see in a shift in distance from the control to higher concentrations of spermin of approximately nm. This shows that in principle it is possible to detect a structure deformation of our biosensor.
Though quantitative evidence is a bit tricky because of the calibration and the fact that one pixel of the pictures equals 101.03nm  a qualitative evidence can be see in a shift in distance from the control to higher concentrations of spermin of approximately nm. This shows that in principle it is possible to detect a structure deformation of our biosensor.


Now that we knew this approach principally works, we decided to take pictures at the TIRF microscope in epifluorescence of a control (the structure immobilized on the surface) and with two different spermine concentrations (1 spermine every 7 bases (1.34 µM spermine) and 1 spermine every 21 bases (0.42 µM spermine)). Every picture was illuminated for 1 sec with the green laser for the green channel and then with the red laser for the red channel for the same time. The graph below now shows the histograms of the distribution of the distance between the maxima of the fitted gaussians.  
Now that we knew this approach principally works, we decided to take pictures at the fluorescence microscope in epifluorescence of a control (the structure immobilized on the surface) and with two different spermine concentrations (1 spermine every 7 bases (1.34 µM spermine) and 1 spermine every 21 bases (0.42 µM spermine)). Every picture was illuminated for 1 sec with the green laser for the green channel and then with the red laser for the red channel for the same time. The graph below now shows the histograms of the distribution of the distance between the maxima of the fitted gaussians in the green and red channel.  


[[Image:Gauss distance hist.png|x450px]]<br>
[[Image:Gauss distance hist.png|x450px]]<br>
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As one can see the distributions look nearly the same for every concentration except for the control. This is due to the small number of points that were measured for this traces. Furthermore the values for each trace seam not to be distributed in a gaussian manner. This maybe underlies the electrostatic repulsion of the arms when the are in close vicinity. Also the distribution reaches to 120 nm. If our structure occupies this conformation the arms are spread very strong which leads to heavy deformations and defects. In addition this wide spread of the distribution also occurs in the control as well as in the two samples with a high concentration of DNA binding molecules. Possible reasons for this artifacts could be a misalignment of the pictures and not accurate enough determination of the spot since we want to measure in the regime of 5 nm which corresponds to a 20th of one single pixel on the detector. So one has to refine the setup and get more values for better statistics to get trustable values of a mean distance of the arms.
As one can see the distributions look nearly the same for every concentration except for the control. This is due to the small number of points that were measured for this traces. Furthermore the values for each trace seam not to be distributed in a gaussian manner. This maybe underlies the electrostatic repulsion of the arms when the are in close vicinity. Also the distribution reaches to 120 nm. If our structure occupies this conformation the arms are spread very strong which leads to heavy deformations and defects. In addition this wide spread of the distribution also occurs in the control as well as in the two samples with a high concentration of DNA binding molecules. Possible reasons for this artifacts could be a misalignment of the pictures and not accurate enough determination of the spot since we want to measure spatial separations of in the regime of 5 nm which corresponds to a 20th of one single pixel on the detector. Also acquisition of uncorrelated spots which belong to different structures might be a problem which can be solved by higher dilution of avidin adaptors and therefor the structure on the slide. So one has to refine the setup and acquire more values for better statistics to get trustable values of a mean distance of the arms.


<h1>Discussion</h1>
<h1>Discussion</h1>

Revision as of 17:12, 2 November 2011

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