Lifetime resolved fluorescence correlation spectroscopy and two-photon spectroscopy of amyloid nanotube bundles
Fluorescence correlation spectroscopy (FCS) has been widely used to investigate molecular dynamics and interactions in biological systems. However, these diffusion based assays currently have a major limitation, which requires that the diffusion coefficients of component species in a sample must be substantially different in order to be resolved. This limitation can be overcome, and the resolution of FCS measurements can be enhanced, by combining FCS measurements with measurements of fluorescence lifetimes. We show that we can dramatically enhance resolution in FCS measurements using global analysis on simultaneously acquired FCS and lifetime data. The method accurately resolves the concentration and diffusion coefficients of multiple sample components, even when their diffusion coefficients are identical, provided that there is a difference in the lifetime of the component species. We show examples of this technique by using both simulations and experiments. It is expected that this method will be of significance for a broad range of researchers studying molecular interactions. In a separate project, a potentially useful amyloid nanotube bundle material from the β-amyloid proteins is studied. We used two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) to investigate the photophysical properties of this material. The emission properties of the bundles are characterized, and their dependence on the moisture level is revealed. The mechanism of the intrinsic fluorescence is discussed to be related to the electron delocalization within the peptides. The spectroscopic and microscopic study of the amyloid nanotube bundles may open up interesting new perspectives in the bio-inspired material science area.