Fluorescence Recovery After Photobleaching (FRAP) with simultaneous Fluorescence Lifetime and time-resolved Fluorescence Anisotropy Imaging (FLIM and tr-FAIM)

We report the simultaneous combination of three powerful techniques in uorescence microscopy: Fluorescence Lifetime Imaging (FLIM), Fluorescence Anisotropy Imaging (FAIM) and Fluorescence Recovery After Photobleaching (FRAP), also called F3 microscopy. An exhaustive calibration of the setup was carried out with several rhodamine 6G (R6G) solutions in water-glycerol and from the combination of the FAIM and FRAP data, the hydrodynamic radius of the dye was directly calculated. The F3 data was analyzed with a home-built MATLAB script, and the setup is currently explored further with Green Fluorescent Protein (GFP). Some molecular dynamic (MD) simulations are currently being run in order to help with the interpretation of the experimental anisotropy data.