Standardization of Fluorophores Using Phase Fluorometry
Measurements of fluorescence lifetimes, like any other analytical procedure, require the use of standards for instrument calibration and comparison. Scattering materials have been widely used in time-resolved fluorometry as standards because, on the nanosecond time scale of fluorescence, their femtosecond “lifetime” is accurately approximated as zero. As is well known (Wahl et al., 1974; Rayner et al., 1976), the use of scattering compounds can introduce artifacts into lifetime determination because the wavelength of emission is necessarily different from that of excitation; e. g., “color effects” in photomultiplier tubes, and other, more subtle optical artifacts (R.B.T. and E.G., unpublished results; Bernard Valeur and David Jameson, personal communication). To minimize these difficulties “standard fluorophores” have long been used, since they permit matching the standard’s emission wavelength with that of the sample (Lako-wicz et al., 1981; F. Castelli, 1985; Kolber and Barkley, 1986). Unfortunately, there is no wide agreement (to better than 5%) on the lifetimes of the standards now in use, even though the precision of current multifrequency phase and pulse instrumentation is perhaps 0.5%. This may be due to differences in the samples themselves, and particularly the concentration of the important quencher, oxygen. Thus a transferable standard might be difficult to specify. Moreover, the dozen or so compounds widely used as standards cover only half the spectral region of interest (250-1000 nm).
KeywordsFluorescence Lifetime Lifetime Data Transferable Standard Wide Agreement Monoexponential Decay
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