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).

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We report silver nanoparticles (Ag NPs) with high stability, sensitivity, and no surface enhanced Raman scattering (SERS) background. The Ag NPs were synthesized via a one-step process with polysodium styrenesulfonate (PSSS) templates, and they could efficiently adsorb polycyclic aromatic molecules via π-π stacking. The adsorption mechanisms and applicability were systematically studied by experimental measurements and theoretical simulations. When the polycyclic aromatic analytes were adsorbed on the PSSS-templated Ag NPs, the vibrations of π-π stacking-bound moieties were attenuated, yet those of the other unbound aromatic moieties increased. Most importantly, when the analytes had more than two π-π stacking binding sites, the PSSS-templated Ag NPs could trap the analytes by focusing through the optical force induced or via the simultaneously formed analyte-Ag NPs aggregates. This afforded high SERS intensity and very low detection limits.

In this paper, we propose a novel and sensitive ratiometric analysis method that uses the fractional intensities of time-resolved fluorescence of genetically encoded fluorescent NADH/NAD+ biosensors, Peredox, SoNar, and Frex. When the conformations of the biosensors change upon NADH/NAD+ binding, the fractional intensities (α i τ i) have opposite changing trends. Their ratios could be exploited to quantify NADH/NAD+ levels with a larger dynamic range and higher resolution versus commonly used fluorescence intensity and lifetime methods. Moreover, only one excitation and one emission wavelength are required for this ratiometric measurement. This eliminates problems of traditional excitation-ratiometric and emission-ratiometric methods. This method could be used to simplify the design and achieve highly sensitive analyte quantification of genetically encoded fluorescent biosensors. Wide potential applications could be developed for imaging live cell metabolism based on this new method.

In this paper, two pyridinium styryl dyes, [2-(4-dimethylamino-phenyl)-vinyl]-1-methylpyridinium iodide (DASPMI), were synthesized and characterized by steady state fluorescence spectroscopy as well as picosecond and femtosecond time-resolved fluorescence spectroscopies. Both dyes exhibit large Stokes shifts and fluorescence decays equivalent to the instrument response function (IRF) standards employed in time-correlated single-photon counting. Due to their styryl and pyridinium moieties, DASPMIs have higher peak fluorescence intensity and shorter excited-state lifetimes than iodide ion-quenched fluorophores. The fluorescence lifetimes of o-DASPMI and p-DASPMI were measured to be 6.6 ps and 12.4 ps, respectively. The fluorescence transients of these DASPMIs were used as the IRFs for iterative reconvolution fitting of the time-resolved fluorescence decay profiles of Rhodamine B (RhB), sulforhodamine B (SRB), and the SRB-SRB2m RNA aptamer complex. The quality of the fits employing the DASPMI-derived IRFs are consistently equivalent to those employing IRFs obtained from light scattering. These results indicate that DASPMI-derived IRFs may be suited for a broad range of applications in time-resolved spectroscopy and fluorescence lifetime imaging microscopy (FLIM), especially in the visible emission range.