Multi-photon excited Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination

Andreas C. Geiger; Casey J. Smith; Garth J. Simpson

doi:10.1117/12.2545908

14 February 2020 Multi-photon excited Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination

Andreas C. Geiger, Casey J. Smith, Garth J. Simpson

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Proceedings Volume 11244, Multiphoton Microscopy in the Biomedical Sciences XX; 112442B (2020) https://doi.org/10.1117/12.2545908
Event: SPIE BiOS, 2020, San Francisco, California, United States

Abstract

Fourier transform fluorescence recovery after photobleaching (FT-FRAP) is proposed and implemented for quantitatively evaluating diffusion and fractional recovery of proteins in complex matrices. Diffusion characterization of proteins is routinely performed for identification of aggregation and for interrogating molecular interactions with excipients. Conventional FRAP is noninvasive, has low sample volume requirements, and can support short measurement times by performing measurements over distances of only a few micrometers. However, conventional measurements are complicated by the need for precise knowledge of the bleach beam profile and potential errors due to sample inhomogeneity. In FT-FRAP, the time-dependent recovery in fluorescence due to diffusion is measured in the spatial Fourier domain, with substantial improvements in the signal-to-noise ratio, mathematical simplicity, representative sampling, and compatibility with multi-photon excitation. A custom nonlinear-optical beam-scanning microscope enabled patterned illumination for photobleaching a sample through two-photon excitation. The fluorescence recovery produced simple single-exponential decays in the spatial Fourier domain. Measurements in the spatial Fourier domain naturally remove bias from imprecise knowledge of the point spread function and reduce measurement variance from inhomogeneity within samples. Comparison between the fundamental FT frequency and higher harmonics has the potential to yield information about anomalous or spatially dependent diffusion with no increase in measurement time. Initial demonstrations of FT-FRAP using patterned illumination are presented, along with a critical discussion of the figures of merit and future developments.

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Andreas C. Geiger, Casey J. Smith, and Garth J. Simpson "Multi-photon excited Fourier-transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination", Proc. SPIE 11244, Multiphoton Microscopy in the Biomedical Sciences XX, 112442B (14 February 2020); https://doi.org/10.1117/12.2545908

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KEYWORDS

Luminescence

Diffusion

Signal to noise ratio

Fourier transforms

Point spread functions

Signal detection

Spatial frequencies

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