Chapter 11: Fluorescence Recovery After Bleaching (FRAP)

Question 1

Define photobleaching

Answer 1

The recovery time a fluorophore requires before it can be re-excited and fluoresce again.

Question 2

What can the measurement of fluorescence recovery be used to determine? Why?

Answer 2

The constant for molecular diffusion.

The mobility of molecules is much greater than singular fluorophore recovery times so the rate at which unbleached molecules can move into the beam path that is responsible for the bleaching can determine the rate of diffusion.

Question 3

Give the equation to determine the constant for molecular diffusion considering (FRAP)

Answer 3

D = diffusion constant
ω = laser beam radius
t_1/2 = time required for intensity to recover to 1/2 initial intensity

Question 4

Give the equation for the recovery function of fluorescence intensity by diffusion

Answer 4

I(t) = fluorescence intensity
I_0 = initial intensity
α = rate constant (based on D)