High speed multispectral fluorescence lifetime imaging
High speed multispectral fluorescence lifetime imaging
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DOI：
10.1364/oe.21.011769
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发表时间：
2013-05-20
期刊：
影响因子：
3.8
通讯作者：
Gerritsen, Hans C.
中科院分区：
文献类型：
--
作者：
Fereidouni, Farzad;Reitsma, Keimpe;Gerritsen, Hans C.
我们报道了一个基于时间选通单光子探测的光谱分辨荧光寿命成像系统，具有200 ps的固定门宽和7个光谱通道。时间门控系统可以在高计数率下操作，但通常具有大的门宽度，并且仅对荧光衰减曲线的一部分进行采样。在这项工作中提出的系统中，使用高速收发器对荧光信号进行采样。进行了误差分析，以表征寿命和光谱检测的性能。门宽度和光谱通道宽度的估计寿命和光谱宽度的准确性的影响进行了描述。整个仪器的性能在高达12 MHz的计数率下进行评估。精确的荧光寿命（误差< 2%）记录在计数率高达5 MHz。这受到PMT性能的限制，而不是电子设备。大型光谱寿命图像集的分析是具有挑战性和耗时的。在这里，我们展示了使用寿命和光谱相量分析图像的成纤维细胞与2个不同的标记组件。相量方法提供了一种快速和直观的方法来分析光谱分辨荧光寿命成像实验的结果。(C)2013年美国光学学会
We report a spectrally resolved fluorescence lifetime imaging system based on time gated single photon detection with a fixed gate width of 200 ps and 7 spectral channels. Time gated systems can operate at high count rates but usually have large gate widths and sample only part of the fluorescence decay curve. In the system presented in this work, the fluorescence signal is sampled using a high speed transceiver. An error analysis is carried out to characterize the performance of both lifetime and spectral detection. The effect of gate width and spectral channel width on the accuracy of estimated lifetimes and spectral widths is described. The performance of the whole instrument is evaluated at count rates of up to 12 MHz. Accurate fluorescence lifetimes (error < 2%) are recorded at count rates as high as 5 MHz. This is limited by the PMT performance, not by the electronics. Analysis of the large spectral lifetime image sets is challenging and time-consuming. Here, we demonstrate the use of lifetime and spectral phasors for analyzing images of fibroblast cells with 2 different labeled components. The phasor approach provides a fast and intuitive way of analyzing the results of spectrally resolved fluorescence lifetime imaging experiments. (C) 2013 Optical Society of America