Improved Estimation of Solute Diffusivity Through Numerical Analysis of FRAP Experiments
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- Irrechukwu, O.N. & Levenston, M.E. Cel. Mol. Bioeng. (2009) 2: 104. doi:10.1007/s12195-009-0042-1
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Accurate determination of solute diffusivities from fluorescence recovery after photobleaching (FRAP) experiments is often hindered by limitations of existing analytical models. This study describes the development and validation of a finite-element-based direct diffusion simulation parameter estimation (DDSPE) method for determining solute diffusivities from FRAP data. The DDSPE method improves on other models by accounting for experimentally measured post-bleaching fluorescence profiles and time-varying boundary conditions, and includes a reaction term to account for the detrimental effects of low level photobleaching produced by image acquisition during recovery. Analyses of simulated FRAP data demonstrate the advantages of this method over common analytical approaches, including a low sensitivity to variations in the spot radius and to the effects of photobleaching during scanning. As an example application, the effects of gel density and dextran size on the diffusivities of fluorescently labeled dextrans (10–250 kDa) in agarose gels (2–6%) were measured via FRAP. As with the simulated data, the DDSPE method was insensitive to spot radius while analytical models were strongly dependent on this experimental parameter. The diffusivities determined by the DDSPE method decreased with increasing solute size and gel density and were in excellent agreement with reference values based on a recent empirical model.