Fig. 1.

Fluorescence perturbation techniques used for effective diffusion measurements, and drawbacks of current analysis methods. a In fluorecence recovery after photobleaching (FRAP) experiments, a small region in the sample is bleached. After bleaching, the diffusion-driven recovery in the bleached region is monitored. b Inverse FRAP (iFRAP) is an experimental mirror image of FRAP: Molecules in a given region are photoconverted and then spread throughout the sample, resulting in the loss of fluorescent signal in the region of photoconversion. c Drawbacks of current analysis methods exemplified with zebrafish development at late blastula stages. Current analysis methods simplify sample geometry, idealise bleaching profiles, or ignore underlying reaction kinetics. d–f Possible relative error in diffusion coefficient estimates that can occur if false assumptions are made about sample geometry (d), bleaching conditions (e), or reaction kinetics (f), respectively. The maximum displayed error was capped to a value of 200%, but can be up to 1000%