Multiparameter single molecule spectroscopy gives insight into the complex photophysics of fluorescence energy transfer (FRET) coupled biosystems

Abstract

Since the discovery of the technique in the early 1990s, single molecule spectroscopy has been used as a powerfultool to investigate and characterize fluorescent molecules, revealing insights into molecular behavior far beyond theinformation content that can be obtained by conventional ensemble studies. Several spectroscopic techniques have beenestablished at the single molecule level, including spectrally resolved fluorescence, fluorescence lifetimeinvestigations, or single molecule Raman measurements. However, the combination of two or more of these spectroscopiesapplied to the same individual molecule in multiparameter approaches yields a deeper understanding of molecular systems.In this contribution, we present our results of combined spectrally-and time-resolved fluorescence microscopy of theintrinsic fluorescence energy transfer (FRET) system of the red fluorescent protein DsRed. Correlating the resultsobtained from the two spectroscopic techniques, we are able to determine all relevant parameters to describe the energytransfer processes within the DsRed system without any further assumptions. We further discuss fluorescence and surfaceenhanced Raman scattering (SERS) spectroscopy of the same individual DsRed unit, which can help to propose mechanisms forphotodegeneration of the distinct chromophores involved. ? 2009 SPIE.