A modified phasor approach for analyzing time-gated fluorescence lifetime images
A modified phasor approach for analyzing time-gated fluorescence lifetime images
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DOI：
10.1111/j.1365-2818.2011.03533.x
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发表时间：
2011-12-01
影响因子：
2
通讯作者：
Gerritsen, H. C.
中科院分区：
文献类型：
--
作者：
Fereidouni, F.;Esposito, A.;Gerritsen, H. C.
荧光寿命成像是一种多功能的工具，允许映射细胞中的生化环境。在各种荧光寿命成像技术中，时间相关单光子计数和时间选通方法已被证明是非常有效和鲁棒的生物样品成像方法。最近，寿命图像的相量表示变得流行，因为它提供了图像的荧光寿命内容的直观图形视图，并且当用于全局分析时，显著提高了寿命分析的整体S/N。与时间相关的单光子计数相比，时间门控方法可以提供更高的计数率（类似于10 MHz），但由于通常使用的门的数量有限，因此以截断和欠采样衰减曲线为代价。这些限制也使时间选通数据的相量分析的实现复杂化。在这项工作中，我们提出并验证了一个理论框架，克服了这些问题。这种改进的方法进行了测试，模拟寿命的图像和细胞。我们证明，这种方法是能够检索两个寿命的时间门控数据，不能使用标准（非全球）拟合技术解决。新方法增加了可以从典型测量中获得的信息，并简化了荧光寿命成像数据的分析。
Fluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, time-correlated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens.Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (similar to 10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells. We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data.