Determination of the halftime of the recovery (t_half)

The halftime (t_half) of recovery is the time from the bleach to the timepoint where the fluorescence intensity reaches the half (I_1/2) of the final recovered intensity (I_E).

Fitting the recovery data to an exponential equation can be used to determine t_half:

If the investigated molecule freely diffuses in the cell or compartment a simple exponential formula should be used:

Where A is the endvalue of the recovered intensity (I_E), t is the fitted parameter and t is the time after the bleaching pulse.

After determination of t by fitting the above equation to the recovery curve the corresponding halftime of the recovery can be calculated with the following formula:

An idealized plot of a FRAP recovery curve.

I_I: initial intensity
I₀: intensity at timepoint t₀ (first postbleach intensity)
I_1/2: half recovered intensity corresponding to t_1/2
       (I_1/2 = (I_E - I₀) / 2)
I_E: endvalue of the recovered intensity
t_half: Halftime of recovery (t1/2 - t0)
Mobile fraction F_m = (I_E - I₀) / (I_I - I₀)
Immobile fraction F_i = 1 - Fm