Multiple time-gate module for fluorescence lifetime imaging
Multiple time-gate module for fluorescence lifetime imaging
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DOI：
10.1366/0003702011952587
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发表时间：
2001-06-01
影响因子：
3.5
通讯作者：
Gerritsen, HC
中科院分区：
文献类型：
--
作者：
de Grauw, CJ;Gerritsen, HC
提出了一种多功能的多时间门荧光寿命采集模块。该模块用作配备脉冲激发的双光子扫描激光显微镜的附加组件，用于荧光寿命成像。它可以通过捕获四个或八个时间门中的强度衰减来记录荧光寿命。该模块允许以高计数率和高效率进行荧光寿命成像。该模块配备了计算机控制的可变延迟线，用于设置门宽度。衰减时间可以在 0.5 ns-1 mu 范围内精确测量。对具有不同（单指数）荧光寿命的多种单一染料溶液进行的荧光寿命成像实验与时间相关单光子计数获得的参考值非常一致。对表现出双指数衰减的测试样本的测量表明，可以高精度地量化两个衰减分量。在包含 6 个不同寿命（范围为 2.8 至 12 ns）的样本中，所有寿命都可以很好地区分。作为一个例子，本文展示了使用羧基荧光素对口腔生物膜进行 pH 成像实验的结果。在这里，荧光寿命成像既用于将短寿命自发荧光与羧基荧光素荧光分开，又用于确定生物膜中的 pH 值。
A versatile multiple time-gate fluorescence lifetime acquisition module is presented. The module is used as an add-on to a two-photon scanning laser microscope equipped with pulsed excitation for fluorescence lifetime imaging. It enables the recording of fluorescence lifetimes by capturing the intensity decay in four or eight time gates. The module allows fluorescence lifetime imaging at high count rates and with a high efficiency. The module is equipped with computer-controlled variable-delay lines for setting the gate widths. Decay times can be accurately measured in the 0.5 ns-1 mus range. Fluorescence lifetime imaging experiments on a number of single dye solutions with different (mono-exponential) fluorescence lifetimes are in excellent agreement with the reference values obtained with time-correlated single photon counting. Measurements on a test specimen exhibiting a bi-exponential decay demonstrate that the two decay components can be quantified with high accuracy. In a specimen containing six different lifetimes, in the range 2.8 to 12 ns, all lifetimes could be well distinguished. As an example, the results of a pH imaging experiment on oral biofilm using carboxyfluorescein are presented. Here, fluorescence lifetime imaging is used both to separate the short-lifetime autofluorescence from the carboxy-fluorescein fluorescence and to determine the pH in the biofilm.