Methods

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Fluorescence recovery after photo bleaching (FRAP)

Fluorescence recovery after photo bleaching (FRAP)

Fluorescence recovery after photo bleaching (FRAP) is sequentially performed in three steps.

The prebleach equilibrium fluorescence signal is measured in a region of interest (ROI) using low laser intensity for a certain period of time.

Sufficiently low laser intensity is preferable to avoid bleaching (1). The ROI is then rapidly bleached with a pulsed IR laser (Ti:Sapphire) with high intensity (2). Finally, the recovery of postbleach fluorescence intensity is detected as a function of time using low laser intensity (3).

Figure 1

The recovery curve can have different shapes, depending on the dynamic of the tracer molecules and the surrounding environment (figure 2). The diffusion coefficient can be found by fitting the recovery curve to an appropriate diffusion model.

Figure 2