Dataset Information

High-Throughput FRAP Analysis of Solute Diffusion in Hydrogels.

ABSTRACT: Increasingly accurate mathematical models have been developed to relate solute and hydrogel properties to solute diffusion coefficients in hydrogels, primarily by comparing solute sizes and hydrogel mesh sizes. Here, we use a standardized, high-throughput method for fluorescence recovery after photobleaching (FRAP) experiments and analysis to characterize the diffusion coefficients of fluorescein, three sizes of FITC-dextran, and three sizes of FITC-conjugated poly(ethylene glycol) (PEG) through 18 structurally varied poly(vinyl alcohol) (PVA) hydrogel formulations. Increasing the hydrogel mesh radii increased the diffusivities of all the tested solutes within the hydrogels. While the diffusivity of FITC-dextrans in hydrogels decreased with increasing solute size, the diffusivity of FITC-PEGs increased with increasing solute size, suggesting that a generalized hydrodynamic radius-based model is not universally applicable for solute diffusion in hydrogels. The high-throughput characterization method for solute diffusion in hydrogels described here facilitates precise hydrogel design for biomedical applications.

SUBMITTER: Richbourg NR

PROVIDER: S-EPMC9122120 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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High-Throughput FRAP Analysis of Solute Diffusion in Hydrogels.

Richbourg Nathan R NR Peppas Nicholas A NA

Macromolecules 20211109 22

Increasingly accurate mathematical models have been developed to relate solute and hydrogel properties to solute diffusion coefficients in hydrogels, primarily by comparing solute sizes and hydrogel mesh sizes. Here, we use a standardized, high-throughput method for fluorescence recovery after photobleaching (FRAP) experiments and analysis to characterize the diffusion coefficients of fluorescein, three sizes of FITC-dextran, and three sizes of FITC-conjugated poly(ethylene glycol) (PEG) through ...[more]

PMID: 35601759

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Project description: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 photobleach beam profile, potential for bias due to sample heterogeneity, and poor compatibility with multiphoton excitation because of local heating. In FT-FRAP with patterned illumination, the time-dependent fluorescence recovery signal is concentrated to puncta in the spatial Fourier domain, 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 photobleach 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 photobleaching 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.

Dataset Information

High-Throughput FRAP Analysis of Solute Diffusion in Hydrogels.

Publications

High-Throughput FRAP Analysis of Solute Diffusion in Hydrogels.

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