Light Quenching of Fluorescence: A New Method to Control the Excited State Lifetime and Orientation of Fluorophores

Abstract

Experimental studies have recently demonstrated that fluorescence emission can be quenched by laser light pulses from modern high-repetition rate lasers, a phenomenon we call "light quenching." In this overview article, we describe the possible effects of light quenching on the steady-state and time-resolved intensity and anisotropy of fluorophores. One can imagine two classes of experiments. Light quenching can occur within the single excitation pulse, or light quenching can be accomplished with a second time-delayed quenching pulse. The extent of light quenching depends on the amplitude of the emission spectrum at the quenching wavelength. Different effects are expected for light quenching by a single laser beam (within a single laser pulse) or for a time-delayed quenching pulse. Depending upon the polarization of the light quenching beam, light quenching can decrease or increase the anisotropy. Remarkably, the light quenching can break the usual z-axis symmetry of the excited state population, and the measured anisotropy (or polarization) depends upon whether the observation axis is parallel or perpendicular to the propagation direction of the light quenching beam. The polarization can increase to unity under selected conditions. Quenching with time-delayed light pulses can result in step changes in the intensity or anisotropy, which is predicted to result in oscillations in the frequency-domain intensity and anisotropy decays. These predicted effects of light quenching, including oscillations in the frequency-domain data, were demonstrated to occur using selected fluorophores. The increasing availability and use of pulsed laser sources requires consideration of the possible effects of light quenching and offers the opportunity for a new class of two-pulse or multiple-pulse time-resolved experiments where the sample is prepared by the excitation pulse and subsequent quenching pulses to modify the excited state population, followed by time- or frequency-domain measurement of the optically prepared excited fluorophores.

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.…”

On the Influence of Higher Excited States on the ISC Quantum Yield of Octa‐aL‐alkyloxy‐substituted Zn‐Phthalocyanine Molecules Studied by Nonlinear Absorption

“…With increasing pump intensities, saturation of the number of excited molecules occurs, due to the saturation of the B-band transition. In addition, taking into account the linear polarization of the pump and of the test beams, an additional saturation effect occurs due to the decrease of light-induced anisotropy with increasing excitation intensities (20). The constant ratio of both intensity dependencies demonstrates that the ISC quantum yield does not depend on the excitation intensity.…”

Two‐Photon STED Spectral Determination for a New V‐Shaped Organic Fluorescent Probe with Efficient Two‐Photon Absorption

“…[19,20] One of the promising methods for the investigation of the excited-state molecular dynamics, including stimulated emission transitions that occur in organic molecules, is a fluorescence quenching methodology described by Lakowicz. [21][22][23] This technique allows modification of the molecular orientational distribution in the excited states, [24,25] creates anisotropic molecular ensembles with specific fluorescence properties, [23] and can reveal the values of one-and two-photon stimulated emission cross sections. [17] The determination of the spectral dependences of one-and two-photon stimulated emission cross sections in combination with comprehensive 2PA investigations is important for further development of high-resolution two-photon fluorescence imaging [26,27] and, in particular, for stimulated emission depletion (STED) microscopy.…”