Expanding the applicability of multiphoton fluorescence recovery after photobleaching by incorporating shear stress in laminar flow.

Abstract

Multi-photon fluorescence recovery after photobleaching (MPFRAP) is a nonlinear microscopy technique used to measure the diffusion coefficient of fluorescently tagged molecules in solution. Previous MPFRAP fitting models calculate the diffusion coefficient in systems with diffusion or diffusion in laminar flow. We propose an MPFRAP fitting model that accounts for shear stress in laminar flow, making it a more applicable technique for in vitro and in vivo studies involving diffusion. Fluorescence recovery curves are generated using high-throughput molecular dynamics simulations and then fit to all three models (diffusion, diffusion and flow, and diffusion and shear flow) to define the limits within which accurate diffusion coefficients are produced. Diffusion is simulated as a random walk with a variable horizontal bias to account for shear flow. Contour maps of the accuracy of the fitted diffusion coefficient as a function of scaled velocity and scaled shear rate show the parameter space within which each model produces accurate diffusion coefficients; the shear-flow model covers a larger area than the previous models. The shear-flow model allows MPFRAP to be a viable optical tool for studying more biophysical systems than previous models.