Keller Scott B, Dudley Jonathan A, Binzel Katherine, Jasensky Joshua, de Pedro Hector Michael, Frey Eric W, Urayama Paul
Department of Physics, Miami University, Oxford, Ohio 45056, USA.
Anal Chem. 2008 Oct 15;80(20):7876-81. doi: 10.1021/ac801252q. Epub 2008 Sep 18.
Time-gated techniques are useful for the rapid sampling of excited-state (fluorescence) emission decays in the time domain. Gated detectors coupled with bright, economical, nanosecond-pulsed light sources like flashlamps and nitrogen lasers are an attractive combination for bioanalytical and biomedical applications. Here we present a calibration approach for lifetime determination that is noniterative and that does not assume a negligible instrument response function (i.e., a negligible excitation pulse width) as does most current rapid lifetime determination approaches. Analogous to a transducer-based sensor, signals from fluorophores of known lifetime (0.5-12 ns) serve as calibration references. A fast avalanche photodiode and a GHz-bandwidth digital oscilloscope is used to detect transient emission from reference samples excited using a nitrogen laser. We find that the normalized time-integrated emission signal is proportional to the lifetime, which can be determined with good reproducibility (typically <100 ps) even for data with poor signal-to-noise ratios ( approximately 20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.
时间选通技术对于在时域中快速采样激发态（荧光）发射衰减很有用。门控探测器与明亮、经济的纳秒脉冲光源（如闪光灯和氮气激光器）相结合，对于生物分析和生物医学应用来说是一种有吸引力的组合。在此，我们提出一种用于寿命测定的校准方法，它是非迭代的，并且不像大多数当前的快速寿命测定方法那样假定仪器响应函数可忽略不计（即激发脉冲宽度可忽略不计）。类似于基于换能器的传感器，来自已知寿命（0.5 - 12纳秒）荧光团的信号用作校准参考。使用快速雪崩光电二极管和GHz带宽数字示波器来检测使用氮气激光器激发的参考样品的瞬态发射。我们发现归一化的时间积分发射信号与寿命成正比，即使对于信噪比很差（约为20）的数据，也能以良好重现性（通常<100皮秒）确定寿命。结果与模拟结果吻合良好。此外，还提出了一种用于荧光寿命成像应用的新时间选通方案。总之，基于校准的方法是在使用时间选通检测和有限脉冲宽度激发的应用中快速测定寿命的一种有价值的分析工具。
