Biomod/2011/TUM/TNT/LabbookA/evaluation of TIRF data in Matlab: Difference between revisions
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Revision as of 07:05, 27 October 2011
In order to analyze the acquired FRET movies and extract accurate FRET efficiencies, a homemade Matlab software was used.
The software automatically picks spots 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.
