Multiphoton-excited Fourier transform fluorescence recovery after photobleaching (FT-FRAP) is implemented for quantitatively evaluating normal and anomalous diffusion and fractional recovery of proteins in complex matrices and heterogeneous samples. Diffusion characterization is routinely performed to quantify the mobility of proteins in cells, pharmacology, and food science. Conventional fluorescence recovery after photobleaching (FRAP) allows for diffusion characterization while being noninvasive and rapid, however conventional point bleach FRAP suffers from low signal-to-noise due to shallow bleach depths, the need for precise knowledge of the photobleaching beam profile for accurate analysis, potential bias from sample heterogeneity, and poor compatibility with multiphoton excitation due to local heating. In pattern illuminated FT-FRAP, a custom nonlinear optical beam-scanning microscope enables a comb bleach profile spread over the field of view. The time-dependent fluorescence recovery is concentrated to points in the spatial Fourier domain leading to a large signal-to-noise enhancement. Analysis in the Fourier domain greatly simplifies the mathematical treatment as the dependence on the bleach profile is removed. The rapid beam scanning is easily compatible with multiphoton excitation which allows for label-free diffusion analysis of proteins through the two-photon excited UV fluorescence of native tryptophan residues. Diffusion at multiple length scales can be quantified using the multiple spatial harmonics of the fluorescence recovery. Spatial-frequency peak shape analysis allows for the simultaneous determination of diffusion at every location in the field of view. Peaks in the spatial Fourier domain are shifted to the origin followed by an inverse Fourier transform to generate fluorescence recover maps. Each pixel was fit to a diffusion model to generate diffusion images.

Diffusion analysis by fourier transform-FRAP with multiphoton-excited patterned illumination

ACS Fall 2021

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