# Biomod/2011/TUM/TNT/Results

(Difference between revisions)
 Revision as of 22:48, 2 November 2011 (view source)← Previous diff Revision as of 23:00, 2 November 2011 (view source)Next diff → Line 148: Line 148:

Fluorescence Tracking

Fluorescence Tracking

- Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determine the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excite the Atto 550 dye and observe at its characteristic wavelength and then excite the Atto 647N dye and observe at its characteristic wavelength. + Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determined the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excited the Atto 550 dye and observed at its characteristic wavelength, subseqeuntly Atto 647N was excited and observed. For the analysis with the homemade matlab script at first we had to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/10/19#Camera_calibration calibrate the cameras]. - For the analysis with the homemade matlab script at first we had to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/10/19#Camera_calibration calibrate the cameras]. + Then the matlab script searched for spots in the green and the red picture and fitted them with a gaussian. The peaks from the green picture then are transfered into the red picture. When there is a matching red spot for the green spot the distance between them is calculated. - Then the matlab script searches for spots in the green and the red picture and fits in an gaussian. The peaks from the green picture then are transfered into the red picture. When there is a matching red spot for the a green spot the distance between them is calculated. + We did those measurements for a control and for two different concentrations of spermine. - We did those measurements for a control and for two different concentrations of spermin. + Quantitative evidence is a bit tricky because of the calibration and the fact that one pixel of the pictures equals 101.03nm. Nevertheless we decided to take pictures in epifluorescence mode of a negative control without DNA binders and with two different spermine concentrations (one spermine every 7 bases and one spermine every 21 bases). 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 (figure 19) shows the histograms of the distribution of the distance between the maxima of the fitted gaussians in the green and red channel. - 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. + [[Image:Gauss distance hist.png|x450px|thumb|Fig. 10 Distance distributions between the fluorescent dyes with varying spermine concentrations]]
- 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. + 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 these 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 they are in close vicinity. Also the distribution reaches up to 120 nm. This is not realistic. Possible reasons for this artifacts could be misalignments of the pictures and not accurate enough determination of the spots since we wanted to measure spatial separations 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. 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. - + - [[Image:Gauss distance hist.png|x450px]]
+ - '''Fig: Histogram of the calculated distances of the arms for control (red), 0.42 µM spermine (blue) and 1.34 µM spermine (green) with a binning of 3 nm. + - ''' + - + - 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. +

Discussion

Discussion

TUM NanU - Home