title: Instrument response standard in time-resolved fluorescence spectroscopy at visible wavelength: quenched fluorescein sodium.
abstract: Time-resolved fluorescence properties of quenched fluorescein sodium, including self-quenching and collisional quenching by iodide, have been studied by using a picosecond time-correlated single-photon counting (TCSPC) apparatus, together with an upconversion spectrophotofluorometer with a time resolution better than 300 fs. The steady-state fluorescence intensity of fluorescein sodium reached the maximum when its concentration was 510 μM with pH > 9. Both the fluorescence intensity and lifetime decreased with increasing concentrations of NaI quencher. When the NaI concentration was 12.2 M, a monoexponential decay with a lifetime as short as 17 ps was exactly determined for the first time using the femtosecond-resolved upconversion system. Picosecond time-resolved fluorescence measurements of circular permuted green and yellow fluorescent proteins (cpGFP and cpYFP) were reported, demonstrating that the fluorescence decay of quenched fluorescein sodium is a better approximation of the instrument response function (IRF) needed for the accurate deconvolution of fluorescence lifetime data, particularly for detectors used in the visible spectral region. We believe that this picosecond lifetime standard will find wide applications in fluorescence lifetime imaging microscopy (FLIM).
authors.affiliation: East China Normal University, State Key Laboratory of Precision Spectroscopy, No. 3663, North Zhongshan Road, Shanghai 200062, P.R. China.
authors.forename: Mengwei
authors.initials: M
authors.lastname: Liu
authors.forename: Menghui
authors.initials: M
authors.lastname: Jia
authors.forename: Haifeng
authors.initials: H
authors.lastname: Pan
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Mengfang
authors.initials: M
authors.lastname: Chang
authors.forename: Hua
authors.initials: H
authors.lastname: Ren
authors.forename: Françoise
authors.initials: F
authors.lastname: Argoul
authors.forename: Sanjun
authors.initials: S
authors.lastname: Zhang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Xu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D004867:Equipment Design; D019793:Fluorescein / Q000737:chemistry*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D017785:Photons; D012015:Reference Standards; D012822:Silicon Dioxide / Q000737:chemistry; D012974:Sodium Iodide / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013995:Time
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D012822:Silicon Dioxide; D012974:Sodium Iodide; D019793:Fluorescein