Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy
Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy
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DOI：
10.1021/ac062160k
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发表时间：
2007-03-01
影响因子：
7.4
通讯作者：
Miura, Atsushi
中科院分区：
文献类型：
--
作者：
Boens, Noel;Qin, Wenwu;Miura, Atsushi
利用单光子计时和多频相位调制荧光仪，以激光为激发源，由9个实验室在20℃的液体溶液中独立测量了一系列具有单指数荧光衰减的荧光团。可作为时间域和频域测量的荧光寿命标准的染料都可以在商业上获得，在测量条件下是光稳定性的，并且可以溶于光谱性质的溶剂(甲醇、环己烷、水)。这些寿命标准是菲、9-氰基菲、9，10-二苯基、N-甲基咔唑、香豆素153、红素B、N-乙酰基-L-色氨酰胺、1，4-二(5-苯并恶唑-2-基)苯、2，5-二苯基恶唑、罗丹明B、Rubrene、N-(3-磺丙基)吖啶和1，4-二苯基苯。在20摄氏度时，荧光寿命从89ps到31.2 ns不等，这取决于荧光染料和溶剂，这是现代皮秒和纳秒或兆赫频域仪器的有用范围。衰减时间与激发波长和发射波长无关。根据染料的结构和溶剂的不同，使用的激发波长从284到575 nm，发射从330到630 nm。这些寿命标准可用于校准或测试时间域和频域仪器的分辨率，或用作消除光电倍增管中颜色效应的参考化合物。用双样本图表进行的统计分析表明，寿命测定不存在实验室偏差。此外，统计检验表明，用时间域和频域荧光法估算的寿命之间有很好的相关性。给出了20种(荧光寿命标准/溶剂)组合的结果汇编综合表格。
A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-L-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 degrees C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and the solvent, the excitation wavelengths used range from 284 to 575 nm, the emission from 330 to 630 nm. These lifetime standards may be used to either calibrate or test the resolution of time- and frequency-domain instrumentation or as reference compounds to eliminate the color effect in photomultiplier tubes. Statistical analyses by means of two-sample charts indicate that there is no laboratory bias in the lifetime determinations. Moreover, statistical tests show that there is an excellent correlation between the lifetimes estimated by the time-domain and frequency-domain fluorometries. Comprehensive tables compiling the results for 20 (fluorescence lifetime standard/solvent) combinations are given.