Calibration Approach for Fluorescence Lifetime Determination for Applications Using Time-Gated Detection and Finite Pulse Width Excitation
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 biomedi...
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Veröffentlicht in: | Analytical chemistry (Washington) 2008-10, Vol.80 (20), p.7876-7881 |
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Format: | Artikel |
Sprache: | eng |
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Zusammenfassung: | 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 |
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ISSN: | 0003-2700 1520-6882 |
DOI: | 10.1021/ac801252q |