THE INTERSECTION OF PHOTOPHYSICS AND SINGLE MOLECULE TRACKING

Single molecule tracking studies through fluorescence time-lapse microscopy and the resulting kinetic that can be determined from such tracking studies present an opportunity to directly observe molecules walking the stochastic path. However, to observe these molecules there must be some label present such as a synthetic fluorophore. These fluorophores have a quantum mechanical nature and when interacting with light can prompt the fluorescence transitions along with spin forbidden transitions to the long-lived triplet state. The transition to the triplet state causes the molecule to temporarily no longer emission of photons, the molecule from here can become permanently non-emitting if it undergoes photo-bleaching. These phenomena need to be probed and measured for the full utility of the fluorophore to be elucidated. A full photophysical characterization is required, the fluorescence spectrum, absorption spectrum, the fluorescent lifetime, fluorescent quantum yield, the intersystem crossing rate, and the phosphorescence rate must be determined. All of these rates are involved in describing the observable phenomenon for an accurate description of the probe for use in biophysical kinetics studied through single molecule tracking. Presented in this dissertation is the construction of a single molecule tracking microscope with time-lapse imaging for the measurement of kinetic studies. Also presented is the instrumental design, construction, and troubleshooting for a confocal fluorescence microscope that can be used to do time-correlated measurements through fluorescence correlation spectroscopy. This microscope facilitates the ability to directly measure the photophysical rates of fluorescence probes. Finally, a study is presented for the full photophysical characterization of single molecule dyes, silicon and germanium rhodamine, highlighting the effect of spin-orbit coupling on the triplet state involved rates. Ultimately the utility of direct observation is reliant on the ability to understand single molecule fluorophores and presented here are methods to accomplish a full photophysical characterization of important single molecule fluorophores.