Mazza Davide, Cella Francesca, Vicidomini Giuseppe, Krol Silke, Diaspro Alberto
Laboratory for Advanced Microscopy, Bioimaging, and Spectroscopy-MicroSCoBiO Research Center, Department of Physics, University of Genoa, Italy.
Appl Opt. 2007 Oct 20;46(30):7401-11. doi: 10.1364/ao.46.007401.
The quantitative analysis of fluorescence perturbation experiments such as fluorescence recovery after photobleaching (FRAP) requires suitable analytical models to be developed. When diffusion in 3D environments is considered, the description of the initial condition produced by the perturbation (i.e., the photobleaching of a selected region in FRAP) represents a crucial aspect. Though it is widely known that bleaching profiles approximations can lead to errors in quantitative FRAP measurements, a detailed analysis of the sources and the effects of these approximations has never been conducted until now. In this study, we measured the experimental 3D bleaching distributions obtained in conventional and two-photon excitation schemes and analyzed the deviations from the ideal cases usually adopted in FRAP experiments. In addition, we considered the non-first-order effects generated by the high energy pulses usually delivered in FRAP experiments. These data have been used for finite-element simulations mimicking FRAP experiments on free diffusing molecules and compared with FRAP model curves based on the ideal bleach distributions. The results show that two-photon excitation more closely fits ideal bleaching patterns even in the event of fluorescence saturation, achieving estimations of diffusion coefficients within 20% accuracy of the correct value.
对诸如光漂白后荧光恢复（FRAP）等荧光扰动实验进行定量分析，需要开发合适的分析模型。当考虑三维环境中的扩散时，对扰动产生的初始条件（即FRAP中选定区域的光漂白）的描述是一个关键方面。尽管众所周知，漂白轮廓近似会导致FRAP定量测量中的误差，但迄今为止从未对这些近似的来源和影响进行过详细分析。在本研究中，我们测量了在传统和双光子激发方案中获得的实验性三维漂白分布，并分析了与FRAP实验中通常采用的理想情况的偏差。此外，我们考虑了FRAP实验中通常施加的高能脉冲产生的非一级效应。这些数据已用于模拟自由扩散分子的FRAP实验的有限元模拟，并与基于理想漂白分布的FRAP模型曲线进行比较。结果表明，即使在荧光饱和的情况下，双光子激发也更接近理想的漂白模式，扩散系数的估计值与正确值的偏差在20%以内。
