University of Lille, Biophotonique Cellulaire Fonctionnelle, Parc de la Haute Borne, 59650 Villeneuve d'Ascq, France.
Cytometry A. 2011 Feb;79(2):149-58. doi: 10.1002/cyto.a.20996. Epub 2010 Dec 2.
We calculate here analytically the performance of the polar approach (or phasor) in terms of signal-to-noise ratio and F values when performing time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) to determine the minimal number of photons necessary for FLIM measurements (which is directly related to the F value), and compare them to those obtained from a well-known fitting strategy using the Least Square Method (LSM). The importance of the fluorescence background on the lifetime measurement precision is also investigated. We demonstrate here that the LSM does not provide the best estimator of the lifetime parameter for fluorophores exhibiting mono-exponential intensity decays as soon as fluorescence background is superior to 5%. The polar approach enables indeed to determine more precisely the lifetime values for a limited range corresponding to usually encountered fluorescence lifetime values. These theoretical results are corroborated with Monte Carlo simulations. We finally demonstrate experimentally that the polar approach allows distinguishing in living cells two fluorophores undetectable with usual time-domain LSM fitting software.
我们在此通过分析来计算在进行时域荧光寿命成像显微镜（FLIM）时，极坐标法（或相位向量法）在信噪比和 F 值方面的性能，以确定 FLIM 测量所需的最小光子数（这与 F 值直接相关），并将其与使用最小二乘法（LSM）的知名拟合策略获得的值进行比较。还研究了荧光背景对寿命测量精度的重要性。我们在此证明，只要荧光背景大于 5%，对于表现出单指数强度衰减的荧光团，LSM 并不能提供寿命参数的最佳估计值。极坐标法确实可以更精确地确定与通常遇到的荧光寿命值相对应的有限范围内的寿命值。这些理论结果得到了蒙特卡罗模拟的验证。我们最后通过实验证明，极坐标法可以在活细胞中区分两种通常使用时域 LSM 拟合软件无法检测到的荧光团。
