Photoswitching Mechanism of Cyanine Dyes

Photoswitchable fluorescent probes have been used in recent years to enable super-resolution fluorescence microscopy by single-molecule imaging.1-6 Among these probes are red carbocyanine dyes, which can be reversibly photoconverted between a fluorescent state and a dark state for hundreds of cycles, yielding several thousand detected photons per switching cycle, before permanent photobleaching occurs.7,8 While these properties make them excel-lent probes for super-resolution imaging, the mechanism by which cyanine dyes are photoconverted has yet to be determined. Such an understanding could prove useful for creating new photoswit-chable probes with improved properties. The photoconversion of red cyanine dyes into their dark states occurs upon illumination by red light and is facilitated by a primary thiol in solution,7,9 whereas agents with a secondary thiol do not support photoswitching. These observations suggest that the reactiv

Photochemical reaction dynamics of 2,2′-dithiobis(benzothiazole): direct observation of the addition product of an aromatic thiyl radical to an alkene with time-resolved vibrational and electronic absorption spectroscopy

The photochemical reaction dynamics of the benzothiazole-2-thiyl (BS) radical, produced by 330 nm ultraviolet photolysis of 2,2′-dithiobis(benzothiazole) (BSSB), are examined on the picosecond time scale. The initial addition product of a thiol–ene reaction between the BS radical and styrene is directly observed by transient vibrational absorption spectroscopy (TVAS). Transient electronic absorption spectroscopy (TEAS) in the ultraviolet and visible spectral regions reveals rapid formation of the ground state BS radical with a time constant of ∼200 fs. The photolytically generated BS radical decays through geminate recombination to the parent molecule BSSB and competitive formation of a BS radical dimer with a rate coefficient of (3.7 ± 0.2) × 1010 M−1 s−1 in methanol, and thereafter (36 ± 1)% of the initially formed BS radicals survive at the longest time delay (1.3 ns). In styrene solution, in contrast to methanol and toluene solutions, kinetic traces of the BS radical show an additional decay with a time constant of 305 ± 13 ps, and a broad band at 345–500 nm grows with the same time constant, suggesting a bimolecular reaction of the BS radical with styrene. The TVAS measurements reveal an absorption band of the ground state BS radical at 1301 cm−1 in toluene solution, and the band decays with a time constant of 294 ± 32 ps in styrene solution. Two product bands grow at 1239 cm−1 and 1429 cm−1 with respective time constants of 312 ± 68 ps and 325 ± 33 ps, and are attributed to the addition product BS–St radical formed from the BS radical and styrene. A bimolecular reaction rate coefficient of kreact = (3.8 ± 0.2) × 108 M−1 s−1 is deduced and 22 ± 1% of the initially formed BS radicals are converted to the BS–St radical in neat styrene solution