Simultaneous measurement of fluorescence anisotropy and translational fluctuations by polarization-modulated MFICS

Abstract

A novel, high signal-to-noise strategy is presented for phase-selective, wavevector- and polarization-resolved fluorescence fluctuation spectroscopy. The approach is based on molecular Fourier imaging correlation spectroscopy (MFICS) and combines polarization- and intensity-modulated photoexcitation with phase-synchronous detection to simultaneously monitor centre-of-mass and slow fluorescence anisotropy fluctuations from a relatively large number (∼10⁶) of fluorescent molecules. The method is demonstrated using DsRed, a tetrameric complex of fluorescent protein subunits, to unambiguously separate signal contributions due to slow optical conformational fluctuations from translational diffusion.

Keywords:

• anisotropy
• fluorescence fluctuations
• forster energy transfer
• phase-selective measurement
• fluorescent protein photodynamics

Acknowledgements

This work is supported by the National Science Foundation (CHE-0303715) and the National Institutes of Health (R01-GM67891).