Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation
Generalization of the polar representation in time domain fluorescence lifetime imaging microscopy for biological applications: practical implementation
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DOI：
10.1111/j.1365-2818.2012.03651.x
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发表时间：
2012-10-01
影响因子：
2
通讯作者：
Heliot, L.
中科院分区：
文献类型：
--
作者：
Leray, A.;Spriet, C.;Heliot, L.
极坐标表示或相量可以快速、直观地指示荧光寿命图像强度衰减中存在的指数数量，越来越多地用于时域荧光寿命成像显微镜实验。时域荧光寿命成像显微镜实验中极坐标的计算涉及多个实验参数（例如仪器响应函数、背景、角频率、时间通道数量），其作用尚未得到详尽研究。在这里，我们从理论上、计算和实验上研究了每个参数对极坐标计算的影响，并提出了参数优化以最小化误差。我们确定了极坐标计算中可能出现的几个错误来源，并提出了适当的修正来弥补它们。例如，我们证明，当时间通道数量较少时，用于积分计算的数值积分方法可能会产生错误。我们报告了理论广义表达式来补偿这些偏差并保持半圆完整性，从而促进使用不同通道数获取的荧光寿命成像显微镜图像之间的比较。这些理论推广表达式最终得到了蒙特卡罗模拟和实验的证实。
The polar representation or phasor, which provides a fast and visual indication on the number of exponentials present in the intensity decay of the fluorescence lifetime images is increasingly used in time domain fluorescence lifetime imaging microscopy experiments. The calculations of the polar coordinates in time domain fluorescence lifetime imaging microscopy experiments involve several experimental parameters (e.g. instrumental response function, background, angular frequency, number of temporal channels) whose role has not been exhaustively investigated. Here, we study theoretically, computationally and experimentally the influence of each parameter on the polar calculations and suggest parameter optimization for minimizing errors. We identify several sources of mistakes that may occur in the calculations of the polar coordinates and propose adapted corrections to compensate for them. For instance, we demonstrate that the numerical integration method employed for integrals calculations may induce errors when the number of temporal channels is low. We report theoretical generalized expressions to compensate for these deviations and conserve the semicircle integrity, facilitating the comparison between fluorescence lifetime imaging microscopy images acquired with distinct channels number. These theoretical generalized expressions were finally corroborated with both Monte Carlo simulations and experiments.