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The postbleach series monitors the dynamics and extent of the fluorescence recovery. The following hints help to improve the accuracy of the recovery detection:
- The acquisition frequency should be adjusted to resolve the dynamic range of the recovery with good temporal resolution (rule of thumb: at least 20 data points during the time required for the half of the recovery).
- Acquisition photobleaching should be minimized to record the recovery dynamics as precisly as possible.
- The ideal postbleach acquisition duration is 10 to 50 times longer than the halftime (Axelrod 1976). In practice initial experiments should be conducted until no noticeable further increase in fluorescence intensity is detected.
- When using FPs the imaging frequency should not be altered during an experiment because the fraction of FPs driven into dark states could be altered complicating the analysis of the data.
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