Measurement of molecular diffusion in solution by multiphoton fluorescencephotobleaching recovery

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique fo r measuring the three-dimensional (3D) mobility of fluorescent molecules wi th 3D spatial resolution of a few microns,A brief, intense flash of mode-lo cked laser light pulses excites fluorescent molecules via multiphoton excit ation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the hig h-intensity focal volume of the illuminating beam, the bleached region is r estricted to a known, three-dimensionally defined volume. Fluorescence in t his focal volume is measured with multiphoton excitation, using the attenua ted laser beam to measure fluorescence recovery as fresh unbleached dye dif fuses in. The time course of the fluorescence recovery signal after photobl eaching can be analyzed to determine the diffusion coefficient of the fluor escent species. The mathematical formulas used to fit MP-FPR recovery curve s and the techniques needed to properly utilize them to acquire the diffusi on coefficients of fluorescently labeled molecules within cells are present ed here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anoma lous subdiffusion model, as well as in aqueous solutions of wild type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2 )s(-1) in excellent agreement with the results of other techniques.