Biomod/2011/TUM/TNT/LabbookA/evaluation of TIRF data in Matlab
In order to analyze the acquired FRET movies and extract accurate FRET efficiencies, a homemade Matlab software was used.
The software automatically picks spots, based on user chosen thresholds 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. Hereby, intensity levels were extracted by fitting (multi-)step funtions.
Calculating accurate FRET
Background corrected intensity levels for the donor ID and acceptor when both dyes were still present were obtained as described above. The true energy transfer is then given by:
where γ is used to correct for differences in efficiencies between donor and acceptor channel as well as for varying quantum yield. It is calculated from the change in donor intensity ΔID and acceptor intensity as follows: