Biomod/2011/TUM/TNT/LabbookA/2011/10/06

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6th Oct 2011

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Single Molecule Fluorescence Microscopy with Sample BM21_172/173

  • The last single molecule fluorescence microscopy measurement on 29th September 2011 resulted in only few detectable FRET events with seemingly randomly distributed FRET efficiencies. Therefore the sample preparation is optimized (Immobilizing structures with biotin oligos for TIRF) and the framerate settings during measurements are modified. The framerate is set to:
    • 50 ms/frame, 500 frames/video (= setting as used in previous experiments)
    • 100 ms/frame, 300 frames/video
    • 500 ms/frame, 200 frames/video
    • 1 s/frame, 100 frames/video

It is expected that an increased excitation time improves signal to noise ratio, as fluctuations should average out.

Results

Image:Histogramm_0.05s_excitationb.tif
Fig.1: Histogram showing number of detected FRET events in intervals of FRET efficiencies; excitation time 0.05s; data from 10 videos


Image:Histogramm_0.1s_excitationb.tif
Fig.2: Histogram showing number of detected FRET events in intervals of FRET efficiencies; excitation time 0.1s; data from 10 videos


Image:Histogramm_0.5s_excitation.tif
Fig.3: Histogram showing number of detected FRET events in intervals of FRET efficiencies; excitation time 0.5s; data from 7 videos


Image:Histogramm_1s_excitation.tif
Fig.4: Histogram showing number of detected FRET events in intervals of FRET efficiencies; excitation time 1s; data from 5 videos

Excitation time [s/frame] Number of videos Number of detected FRET events
0.05 10 40
0.1 10 64
0.5 7 9
1 5 6

As the table above shows the number of detected FRET events was more than 50% higher in this experiment, when the excitation time was increased from 0.05 s to 0.1s. When the excitation time was set to 0.5 s or 1 s, however, only few FRET events could be detected.