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Depending on the experiment there are several data evaluation steps which have to be carried out before meaningful results can be achieved:
- Alignment of the images (only necessary if the regions of interest moved over time).
- Fluorescence intensity quantification (obtaining the raw data)
- Background subtraction
- Correct for laser fluctuations, photobleaching during acquisition (postbleach) and total fluorescence loss caused by the bleaching step
- Normalization
- Mobile/immobile fraction
- T½ halftime of the equilibration of bleached and unbleached molecules
- Theoretical models to additionally determine binding characteristics of the analysed molecule
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