title: Calibration approach for fluorescence lifetime determination for applications using time-gated detection and finite pulse width excitation.
abstract: 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 <100 ps) even for data with poor signal-to-noise ratios ( approximately 20). Results are in good agreement with simulations. Additionally, a new time-gating scheme for fluorescence lifetime imaging applications is proposed. In conclusion, a calibration-based approach is a valuable analysis tool for the rapid determination of lifetime in applications using time-gated detection and finite pulse width excitation.
authors.affiliation: Department of Physics, Miami University, Oxford, Ohio 45056, USA.
authors.forename: Scott B
authors.initials: SB
authors.lastname: Keller
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Dudley
authors.forename: Katherine
authors.initials: K
authors.lastname: Binzel
authors.forename: Joshua
authors.initials: J
authors.lastname: Jasensky
authors.forename: Hector Michael
authors.initials: HM
authors.lastname: de Pedro
authors.forename: Eric W
authors.initials: EW
authors.lastname: Frey
authors.forename: Paul
authors.initials: P
authors.lastname: Urayama
mesh_terms: D002138:Calibration; D005453:Fluorescence; D012015:Reference Standards; D013997:Time Factors