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bioRxiv · 10.1101/2020.03.13.990937

Anomalous Diffusion Characterization by Fourier-Transform FRAP with Patterned Illumination

Geiger, A. C.·Smith, C. J.·Takanti, N.·Harmon, D. M.·Carlsen, M. S.·Simpson, G. J.
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Abstract

Fourier transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination is theorized and demonstrated for quantitatively evaluating normal and anomalous diffusion. Diffusion characterization is routinely performed to assess mobility in cell biology, pharmacology, and food science. Conventional FRAP is noninvasive, has low sample volume requirements, and can rapidly measure diffusion over distances of a few micrometers. However, conventional point-bleach measurements are complicated by signal-to-noise limitations, the need for precise knowledge of the bleach beam profile, potential for bias due to sample heterogeneity, and poor compatibility with multi-photon excitation due to local heating. In FT-FRAP with patterned illumination, the time-dependent fluorescence recovery signal is concentrated to puncta in the spatial Fourier domain through patterned bleaching, with substantial improvements in signal-to-noise, mathematical simplicity, representative sampling, and multiphoton compatibility. A custom nonlinear-optical beam-scanning microscope enabled patterned illumination for photobleaching through two-photon excitation. Measurements in the spatial Fourier domain removed dependence on the bleach profile, suppressing bias from imprecise knowledge of the point spread function. For normal diffusion, the fluorescence recovery produced a simple single-exponential decay in the spatial Fourier domain, in excellent agreement with theoretical predictions. Simultaneous measurement of diffusion at multiple length scales was enabled through analysis of multiple spatial harmonics of the bleaching pattern. Anomalous diffusion was characterized by FT-FRAP through a nonlinear fit to multiple spatial harmonics of the fluorescence recovery. Constraining the fit to describe diffusion over multiple length scales resulted in higher confidence in the recovered fitting parameters. Additionally, phase analysis in FT-FRAP was shown to inform on flow/sample translation. Statement of SignificanceFourier transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination greatly improves the accuracy of diffusion assessments and simultaneously accesses information on both normal and anomalous diffusion in a single experiment.

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Geiger, A. C., Smith, C. J., Takanti, N., Harmon, D. M., Carlsen, M. S., Simpson, G. J.. 2020-03-13. Anomalous Diffusion Characterization by Fourier-Transform FRAP with Patterned Illumination. https://doi.org/10.1101/2020.03.13.990937

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