Theory for two-photon excitation in pattern photobleaching with evanescent illumination.

Although the translational diffusion coefficients of proteins embedded in, or tightly bound to, natural and artificial cell membranes have been extensively studied, very little is known about the diffusion of proteins weakly bound to membranes. One method for measuring the diffusion coefficients of weakly bound species is to combine evanescent interference patterns with fluorescence pattern photobleaching recovery (TIR-FPPR). However, this technique is intrinsically limited in that the maximum post-bleach fluorescence change, normalized to the pre-bleach fluorescence, is 0.22. This limitation severely restricts the precision of the data and the applicability of the method. In the theoretical work described herein, it is shown that the maximum fractional fluorescence change in TIR-FPPR with two-photon bleaching and observation would be 0.36. Thus, the use of two-photon excitation in TIR-FPPR would significantly improve the signal-to-noise ratio and would provide more precise measurements of the translational diffusion of proteins weakly bound to surfaces.