Single-molecule fluorescence intensity trajectories contain rich information about the molecular dynamics of protein conformational changes, DNA-protein interactions, and protein-protein interactions. However, the typical dynamic range of the single-molecule trajectories has only been from the sub-millisecond to second-and-longer. We have developed single-molecule spectroscopic capabilities to measure the single-molecule fluorescence lifetime trajectories, extending the time scale to the nanosecond regime. At the same time, we have initiated methods to obtain single molecule fluorescence anisotropy trajectories at the nanosecond-to-second time-scale and to achieve a nanoscale topographic characterization in correlations with single-molecule spectroscopy.
Single-Molecule Time-Resolved Fluorescence Anisotropy: Molecule rotational motion rates are dependent upon molecular hydrodynamic shapes and masses that can be changed by molecular interactions and complex formation/dissociation. Measuring the time-dependent anisotropy of a site-specifically labeled dye molecule would be a means of probing the conformational motions of the labeled domain of the protein. Because the time-scale for dye molecule spinning is in the sub-nanosecond range, the nanosecond-to-microsecond protein conformational motions can be differentiated and probed by measuring the time-dependent anisotropy of the dye molecules in this longer time regime. The results will be observed as changes in the fluorescence polarization anisotropy in time. This technique allows us to measure the single-molecule conformational changes as well as the substrate-enzyme complex formation, reaction, and dissociation in real time.
AFM-enhanced optical spectroscopic microscopy: We have been working to combine AFM and single-molecule microscopy in one instrument in order to obtain molecular scale topographic information and the correlated spectroscopic information for the same single-molecule reaction system. This approach enhances research in our protein conformational and enzymatic reaction dynamics project. Single-molecule dynamics have been "traditionally" studied using fluorescence spectroscopy that probes the overall molecular or active site conformational fluctuations and the excited-state lifetimes. Vibrational-mode resolved single-molecule dynamics has never been conducted due to the lack of vibrational specificity of fluorescence. Quite conceivably, Raman spectroscopy, including surface-enhanced Raman spectroscopy (SERS), could provide rich information on vibrational relaxation, vibrational coupling, chemical bond formation/dissociation, and mode-specific nuclear displacements. Based on the near-field enhancement at the metallic AFM tip, we have experimentally demonstrated AFM tip enhanced fluorescence lifetime imaging microscopy (AFM-FLIM) and currently working on demonstrating the AFM tip enhanced surface enhanced Raman spectroscopy (AFM-SERS) imaging guided by finite element method (FEM) computational simulation of the near-field electronmagnetic field distributions.
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