Analysis of Postphotoactivation Scanning Diffusion Profiles for Multiple Species with Distributed Diffusion Coefficients

Abstract

Postphotoactivation scanning (PPS) is a method for quantifying diffusion coefficients of particles in simple and complex materials (e.g., hydrogels) over length scales ∼100μm-mm (Geonnotti et al., 2008). Diffusing particles are labeled with caged fluorophore, and a slit-shaped region of sample is exposed to UV to generate a line of fluorescence. A high-resolution scanner quantifies intensity profiles as particles diffuse out from the fluorescent region over time; these are fit to the solution of the diffusion equation to obtain the diffusion coefficient. We use this technique to measure mobility of HIV-like liposomes. Here, we describe a novel method for analyzing PPS profiles for multiple diffusing species with a distribution (α) of diffusion coefficients (D). To determine α(D), we generated sets of diffusion profiles for a discretized range of D by numerically solving the diffusion equation using the experimental initial condition and assuming given D. We computed net diffusion profiles resulting from the sums of profiles with distribution α. We used an optimization scheme to deduce α(D) that minimized the squared difference of observed and computed profiles. The method was validated using simulated and experimental data. Experimental results for fluorescein diffusing in PBS (D=4.3×10−6 cm2/s) are similar to literature values. We also measured diffusion coefficients of solutions of labeled ∼100nm liposomes (D=3.2±2×10−8 cm2/s, n=4). We were able to resolve 2 distinct peaks in α corresponding to the D of liposomes and of free label. Measured values for diffusion coefficients of liposomes are similar to those predicted by Stokes-Einstein (D=3.9±1×10−8 cm2/s). We are using the technique to analyze interactions of HIV-like liposomes and anti-HIV antibodies. The method can be applied to describe diffusion of multiple species within complex materials. [Supported by Duke CFAR and NIH AI48103]