■光漂白后的多光子荧光恢复(MPFRAP)是一种非线性显微镜技术,用于测量荧光标记的分子在溶液中的扩散系数。以前的MPFRAP拟合模型计算在层流中具有扩散或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,使其成为涉及扩散的体外和体内研究的更适用的技术。
■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)来定义产生精确扩散系数的极限。扩散被模拟为具有可变水平偏置的随机游走,以考虑剪切流。
■拟合扩散系数的精度作为缩放速度和缩放剪切速率的函数的轮廓图显示了每个模型产生准确扩散系数的参数空间;剪切流模型比以前的模型覆盖更大的面积。
■剪切流模型使MPFRAP成为研究比以前模型更多的生物物理系统的可行光学工具。
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.