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Appl Spectrosc. 2016 Jul;70(7):1195-201. doi: 10.1177/0003702816652363. Epub 2016 May 26.

Using Pyridinium Styryl Dyes as the Standards of Time-Resolved Instrument Response.

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State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai, China.
State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai, China
JILA, National Institute of Standards and Technology (NIST), University of Colorado, Boulder, Colorado, USA.


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.


Fluorescence quenching; instrument response function (IRF); pyridinium styryl dyes; time-resolved fluorescence


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