Resolving inhomogeneity using lifetime-weighted fluorescence correlation spectroscopy.
Fluorescence correlation spectroscopy (FCS) was extended by incorporating information of the fluorescence lifetime. This new experimental approach, called lifetime-weighted FCS, enables us to observe fluorescence lifetime fluctuations in the nano- to millisecond time region. The potential of this method for resolving inhomogeneity in complex systems was demonstrated. First, by measuring a mixture of two dye molecules having different fluorescence lifetimes, it was shown that the lifetime-weighted correlation deviates from the ordinary intensity correlation only when the system is inhomogeneous. This demonstrated that lifetime-weighted FCS is capable of detecting inhomogeneity in an ensemble-averaged fluorescence decay profile without any a priori knowledge about the system. Second, we applied this method to a dye-labeled polypeptide, a prototypical model of complex biopolymers. It was found that the ratio between the lifetime-weighted and ordinary intensity correlation changes with change of the environment around the polypeptide. This result was interpreted in terms of environment-dependent conformational inhomogeneity of the polypeptide. Delay time dependence of the ratio was found to be constant from ∼1 μs to several milliseconds, indicating that the observed inhomogeneity is persistent in the measured time scale. In combination with fluorescence intensity correlation, lifetime-weighted FCS allows us to examine conformational fluctuations of complex systems in the time region from nano- to milliseconds, being free from the translational diffusion signal.
Ishii
Kunihiko
K
Molecular Spectroscopy Laboratory, Advanced Science Institute (ASI), RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan.
Tahara
Tahei
T
D005456:Fluorescent Dyes / Q000737:chemistry*; D008968:Molecular Conformation; D010455:Peptides / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013997:Time Factors
D005456:Fluorescent Dyes; D010455:Peptides