Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy

Noël Boens, Wenwu Qin, Nikola Basarić, Johan Hofkens, Marcel Ameloot, Jacques Pouget, Jean Pierre Lefèvre, Bernard Valeur, Enrico Gratton, Martin VandeVen, Norberto D. Silva, Yves Engelborghs, Katrien Willaert, Alain Sillen, Garry Rumbles, David Phillips, Antonie J.W.G. Visser, Arie Van Hoek, Joseph R. Lakowicz, Henryk MalakIgnacy Gryczynski, Arthur G. Szabo, Don T. Krajcarski, Naoto Tamai, Atsushi Miura

Research output: Contribution to journalArticlepeer-review

341 Scopus citations


A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 °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, AT-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 °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.

Original languageEnglish
Pages (from-to)2137-2149
Number of pages13
JournalAnalytical Chemistry
Issue number5
StatePublished - 5 Mar 2007


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