High-performance time-resolved fluorescence by direct waveform recording
High-performance time-resolved fluorescence by direct waveform recording
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DOI：
10.1063/1.3480647
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发表时间：
2010-10-01
影响因子：
1.6
通讯作者：
Thomas, David D.
中科院分区：
文献类型：
--
作者：
Muretta, Joseph M.;Kyrychenko, Alexander;Thomas, David D.
我们描述了一种高性能的时间分辨荧光（HPTRF）光谱仪，大大提高了精确和准确的亚纳秒分辨荧光发射波形可以响应于脉冲激发获得的速率。该仪器的主要特点是一个强烈的（1 μ J/脉冲），高重复率（10 kHz），和短（1 ns半高全宽）激光激发源和瞬态数字转换器（0.125 ns每个时间点），记录一个完整的和准确的荧光衰减曲线的每个激光脉冲。对于含有几纳摩尔染料的典型荧光样品，响应于每0.1ms的单个激光脉冲，可以获得具有约100的信号/噪声的波形，比时间相关单光子计数的常规方法快至少10（5）倍，对于短至100 ps的寿命具有相同的准确度和精度。使用标准的单寿命样品，检测到的信号具有极高的可重复性，波形精度和线性度在单脉冲实验的1%误差内。在0.1秒（1000个脉冲）的HPTRF仪器获得的波形有足够的精度来分析两个样品具有不同的寿命，解决次要成分的寿命和摩尔分数方面的高精度。该仪器使一类新的高通量时间分辨荧光实验成为可能，这些实验对于生物学应用（包括瞬态动力学、多维荧光和微孔板格式）应该特别强大。(C)2010年美国物理学会。[doi：10.1063/1.3480647]
We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1 mu J/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 10(5) times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats. (C) 2010 American Institute of Physics. [doi:10.1063/1.3480647]