University of Rochester, Department of Biomedical Engineering, Rochester, New York, United States.
Manhattan College, Department of Physics, Riverdale, New York, United States.
J Biomed Opt. 2023 Jul;28(7):076502. doi: 10.1117/1.JBO.28.7.076502. Epub 2023 Jul 21.
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 and 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.
多光子荧光漂白后恢复（MPFRAP）是一种非线性显微镜技术，用于测量溶液中荧光标记分子的扩散系数。以前的 MPFRAP 拟合模型计算具有扩散或层流扩散的系统中的扩散系数。
我们提出了一种 MPFRAP 拟合模型，该模型考虑了层流中的剪切应力，使其成为更适用于涉及扩散的 和 研究的技术。
使用高通量分子动力学模拟生成荧光恢复曲线，然后将其拟合到所有三个模型（扩散、扩散和流动以及扩散和剪切流动）中，以确定能够产生准确扩散系数的范围。扩散模拟为具有可变水平偏差的随机游动，以考虑剪切流动。
作为标度速度和标度剪切率函数的拟合扩散系数准确性的等高线图显示了每个模型产生准确扩散系数的参数空间；剪切流动模型覆盖的区域大于以前的模型。
剪切流动模型使 MPFRAP 成为一种可行的光学工具，可用于研究比以前的模型更多的生物物理系统。
