Improved Estimation of Solute Diffusivity Through Numerical Analysis of FRAP Experiments
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.
KeywordsAgarose Dextran Diffusion Finite element Parameter estimation Transport
We are grateful to Johnafel Crowe for assistance with confocal microscopy, to Dr. Wei Sun for assistance with development of the COMSOL model, and to Dr. M. Loraine Lowder for help with MATLAB programming and for fruitful discussions. This work was supported by the Arthritis Foundation through an Arthritis Investigator Award and by the ERC program of the National Science Foundation under award number EEC-9731643 (Georgia Tech/Emory Center (GTEC) for the Engineering of Living Tissue).
- 3.Bert, J. L., R. H. Pearce, J. M. Mathieson, and S. J. Warner 1980 Characterization of collagenous meshworks by volume exclusion of dextrans. Biochem J 191:761–768.Google Scholar
- 12.Lopez, A., L. Dupou, A. Altibelli, J. Trotard, and J. F. Tocanne 1988 Fluorescence recovery after photobleaching (frap) experiments under conditions of uniform disk illumination. Critical comparison of analytical solutions, and a new mathematical method for calculation of diffusion coefficient d. Biophys J 53:963–970.CrossRefGoogle Scholar