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The imaging in a FRAP experiment generally consists of 3 steps:
- The sample is first imaged at low intensity illumination in a prebleach series (usually about 10 images) to measure the fluorsecence equilibrium before disturbance.
- The second step is to bleach one or more spots or regions of interest (ROI) with high intensity illumination to disturb the fluorescence equilibrium.
- In the last imaging step a series of postbleach images is acquired to record the fluorescence recovery kinetics.
Finally several data evaluation steps reveal mobility-related parameters of the molecule of interest.
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