Development of Spectral Imaging Microscope for Single Molecule Studies in Complex Biological Systems

Imaging techniques, such as microscopy have become workhorse methodologies in virtually all-scientific disciplines. As such, there is a continuing drive to increase the amount of information density while maintaining the capability for high resolution and high acquisition rates. Most high-resolution techniques fall into one of two categories: high quantum efficiency time-resolved single photon counting, or much lower efficiency spectroscopies using dispersion type monochrometers/spectrometers. In fact, a combination of these techniques has the potential to provide us with greater information density like the temporal behavior and the point of origin within a 3-D sample of each photon in a scanning format. This approach has been advanced recently using single-photon counting techniques coupled with high efficiency optics providing sub-ns time resolution and also limited photonic energy resolution. By further extending these techniques to the single molecule level, where the underlying photophysics of the probe fluor are carefully characterized, the quantum mechanical nature of the flour can be used to statistically analyze the photon stream revealing the underlying physical and chemical processes within the system of interest with a resolution not previously obtained. Unlike traditional spectroscopies, the sub-ensemble nature of the single molecule experiment is uniquely sensitive to rare events and random fluctuations, which are otherwise washed out in bulk measurements due to their low relative probability and the use of experimental averaging. Single molecule Fluorescence (FL) techniques are now being applied in biology, but photostability and poor s/n prohibit correlations between temporal and spatial information. Simultaneous application of FL and Raman imaging spectroscopies has the potential to overcome these obstacles paving the way for detailed investigations of many biological processes. Time-resolved FL and Raman measurements are usually considered to be orthogonal techniques, especially when carried out at high resolution, where few species are being probed. A combination of these techniques becomes possible at the single probe level. With this approach the temporal, spectroscopic and chemical signatures of individual species can be observed, probing their local environment with nanometer spatial resolution. This technique promises to be a powerful tool for the biological sciences capable of investigating a broad range of properties, which includes: folding kinetics, translational diffusion (transduction), binding and reorganization, chemical and rotational dynamics (thermodynamics), solvent dynamics and viscosity, charge transfer, small molecule or ion/proton exchange (signal transduction), and electronic state dynamics