Light Quenching

  Time-resolved fluorescence is routinely measured using one pulse to excite the sample, followed by measurement of the time-dependent emission. In Light Quenching one uses additional pulses following the excitation pulse to modify the excited state population. This occurs upon illumination with longer wavelength non-absorbing light which depletes part of the excited population by stimulated emission.

The fluorophores are not actually quenched, but appear to be quenched because we observed the residual population at right angles to the "quenching beam". The "quenched" part of the emission travels parallel to the quenching beam and is not observed. In time-resolved Light Quenching experiments we observe the emission before and after the quenching pulse. The instantaneous change in the intensity and/or anisotropy decays reflects in frequency-domain as characteristic oscillations.

Light Quenching offers a unique opportunity to control an excited state population and orientation of fluorophores. In presence of Light Quenching we observed fluorescence anisotropies above 0.4 and below -0.2.

We demonstrated that Light Quenching can be used to:

• Study the spectral relaxation of solvent-sensitive fluorophores
• Eliminate unwanted fluorescent species from the mixture of fluorophores
• Increase the spatial resolution in far-field fluorescence microscopy
• Quench the fluorescence near the glass surface in total internal reflection excitation

We also developed a generalized theory of fluorescence anisotropy in preserve of LQ
 

Figure: Light quenching with parallel excitation and quenching pulses. Excited state population without light quenching (top), with time-coincident or one-pulse light quenching (middle) and with time-delayed light quenching (bottom)

References:

1. Stimulated Emission on Microscopic Scale: Light Quenching of Pyridinium 2 Using a Ti:Sapphire Laser. Hell, S.W., Schrader, M., Bahimann, K., Meinecke, F., Lakowicz, J.R. and Gryczynski, I. (1995). J. Microscopy, 180(2), RP1-RP2.
2. Wavelength-Selective Light Quenching of Biochemical Fluorophores. Gryczynski, I., Kusba, J. and Lakowicz, J.R. (1997) J. Biomed. Optics, 2(1):80-87.
3. Effect of Fluorescence Quenching by Stimulated Emission on the Spectral Properties of a Solvent-Sensitive Fluorophore. Gryczynski, I., Kusba, J., Gryczynski, Z., Malak, H. and Lakowicz, J.R. (1996) J. Phys. Chem., 100(24):10135-10144.
4. On the Possibility of Evanescent Wave Excitation Distal from a Solid-Liquid Interface Using Light Quenching. Lakowicz, J.R., Gryczynski, Z., and Gryczynski, I. (1996). Photochem. Photobiol., 64(4):636-641.
5. Light Quenching of Pyridine 2 Fluorescence with Time-Delayed Pulses. Gryczynski, I., Hell, S.W., and Lakowicz, J.R. (1997). 66:13-24.
6. Effects of Light Quenching on the Emission Spectra and Intensity Decays of Fluorophore Mixtures. Gryczynski, I., Kusba, J., and Lakowicz, J. R. (1997). J. Fluoresc. 7(3):167-183.
7. Anisotropy Spectra of the Solvent-Sensitive Fluorophore 4-Dimethylamino-4'-Cyanostilbene in the Presence of Light Quenching. Gryczynski, I., Kusba, J., Gryczynski, Z., Malak, H., and Lakowicz, J. R. (1998). J. Fluoresc. 8:253-261. (abstract)
8. Fluorescence Anisotropy Controlled by Light Quenching. Gryczynski, I., Gryczynski, Z., and Lakowicz, J. R. (1998). Photochem. Photobiol. 67(6):641-646. (abstract)
9. Definition and properties of the emission anisotropy in the absence of cylindrical symmetry of the emission field: Application to the light quenching experiments, Kusba, J., and Lakowicz, J. R. (1999). J. Chem. Phys. 111:89-99.