Sullivan Kelley D, Brown Edward B
Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, USA.
Phys Rev E Stat Nonlin Soft Matter Phys. 2011 May;83(5 Pt 1):051916. doi: 10.1103/PhysRevE.83.051916. Epub 2011 May 16.
Multiphoton fluorescence recovery after photobleaching (MP-FRAP) is a laser microscopy technique used to measure diffusion coefficients of macromolecules in biological systems. The three-dimensional resolution and superior depth penetration within scattering samples offered by MP-FRAP make it an important tool for investigating both in vitro and in vivo systems. However, biological systems frequently confine diffusion within solid barriers, and to date the effect of such barriers on the measurement of absolute diffusion coefficients via MP-FRAP has not been studied. We have used Monte Carlo simulations of diffusion and MP-FRAP to understand the effect of barriers of varying geometries and positions relative to the two-photon focal volume. Furthermore, we supply ranges of barrier positions within which MP-FRAP can confidently be employed to measure accurate diffusion coefficients. Finally, we produce two new MP-FRAP models that can produce accurate diffusion coefficients in the presence of a single plane boundary or parallel infinite plane boundaries positioned parallel to the optical axis, up to the resolution limit of the multiphoton laser scanning microscope.
多光子光漂白后荧光恢复（MP-FRAP）是一种激光显微镜技术，用于测量生物系统中大分子的扩散系数。MP-FRAP在散射样品中提供的三维分辨率和出色的深度穿透能力，使其成为研究体外和体内系统的重要工具。然而，生物系统常常将扩散限制在固体屏障内，迄今为止，此类屏障对通过MP-FRAP测量绝对扩散系数的影响尚未得到研究。我们利用扩散和MP-FRAP的蒙特卡罗模拟，来了解不同几何形状和相对于双光子焦体积的位置的屏障的影响。此外，我们给出了屏障位置范围，在该范围内可以放心地使用MP-FRAP来测量准确的扩散系数。最后，我们提出了两个新的MP-FRAP模型，在存在与光轴平行的单个平面边界或平行无限平面边界的情况下，直至多光子激光扫描显微镜的分辨率极限，这两个模型都能产生准确的扩散系数。
