Effect of Fluorescence Quenching by Stimulated Emission on the Spectral Properties of a Solvent-Sensitive Fluorophore

Abstract

We examined the emission spectra and wavelength-dependent anisotropies of the solvent-sensitive fluorophore 4-(dimethylamino)-4′-cyanostilbene (DCS) under condition of fluorescence quenching by stimulated emission. The sample was illuminated with a train of 10 ps pulses at 285 nm, and a train of stimulating pulses at 570 nm which were delayed by a time t d relative to the excitation. Stimulated emission of DCS was demonstrated to occur by observation of gain in the long-wavelength beam. Illumination on the long-wavelength side of the emission spectrum with the long-wavelength time-delayed pulses resulted in a blue shift of the emission spectrum, and a progressive decrease of the emission anisotropy as the observation wavelength increased toward the stimulating wavelength. The spectral shifts and wavelength-dependent anisotropies of DCS were more pronounced in more viscous solvents where spectral relaxation is incomplete during the excited state lifetime. Light quenching of DCS in a low-viscosity solvent revealed no spectral shifts or wavelengthdependent anisotropies. Control measurements using acridine orange, which is relatively insensitive to solvent polarity, did not show any spectral shift or wavelength-dependent anisotropy with light quenching. The data for DCS can be explained by the presence of a time-dependent spectral shift and wavelength-selective quenching of the longer wavelength emission. In this model the relaxed state is formed following excitation of the unrelaxed state, and the relaxed state is preferentially quenched by long-wavelength illumination. Comparison of the data with model calculations indicates the presence of at least two spectral relaxation times. These results demonstrate that light quenching by stimulated emission acts selectively based on overlap of the stimulating wavelength with the emission spectrum. Observation of the emission spectrum in the presence of time-delayed and power-controlled long-wavelength pulses can be used to study time-dependent excited state processes.

Light quenching of pyridine2 fluorescence with time-delayed pulses

“…The emission spectra are nearly identical in the absence and presence of light quenching. We recognize that the presence or absence of spectral shifts with light quenching may provide information on the rates of solvent relaxation, and such shifts have been observed [32]. In other studies of light quenching of other fluorophores we showed that the extent of quenching was proportional to the amplitude of the emission spectrum at the quenching wavelength.…”

Fluorescence Spectral Properties of 2,5-Diphenyl-1,3,4-oxadiazole with Two-Color Two-Photon Excitation

“…26,[39][40][41] This phenomenon, which we call light quenching, has already been applied to fluorescence microscopy. 28,42 In ref 42 the authors describe the use of a modulated signal when the light quenching pulses are at a repetition frequency offset from the excitation pulse rate.…”

“…25,26 Unless stated otherwise, both beams were vertically polarized. Spatial and temporal overlap was accomplished by maximizing light quenching of Pyridine 2 in methanol, which can be accomplished with 380 and 760 nm, respectively.…”

Determination of the Formation of Dark State via Depleted Spontaneous Emission in a Complex Solvated Molecule

“…In order to get full information about the ultrafast dynamics, we here develop the isotropic (54.7°) and anisotropic (parallel and vertical) femtosecond fluorescence depletion methods, where the polarizations of dumping beams with respect to that of pumping beam are selected by rotating a zero-level half-wave plate to obtain isotropic (54.7°) and anisotropic (parallel and vertical) results, respectively. The time-resolved anisotropic decay r(t) was calculated from the decay curves under the condition of parallel polarizations (I || ) and perpendicular polarizations (I ⊥ ) of dumping beam relative to the polarization of pumping beam according to eq 3: 33,35,36 The factor G accounts for the difference in sensitivities for the detection of signal in the perpendicular-and parallelpolarized configurations, where G (G ) I ⊥ /I || , when the excitation is vertically polarized) is estimated about 1.02. The instrument response function is determined according to the 1 + 1′ two photon excited fluorescence method as described before, 10,11,34 in which, the IRF (instrumental response function) up to 90 fs is achieved.…”