Fluorescence Quantification by Photon Statistics: From Objective Characterization to Application

Quantitative information is key to unravel molecular processes in all fields of research. Counting by Photon Statistics (CoPS) is a single molecule technique that provides such quantification for fluorescent species. CoPS exploits the photon antibunching effect to infer the number of independent emitters and their molecular brightness from multiple photon detection events. I laid the foundation for high quality results by improving the microscope detection efficiency more than threefold compared to earlier measurements. Using both simulations and experiments with defined, DNA-based probes, I investigated the critical interplay of fluorophore properties and analysis parameters. I discovered that measurements at high molecular brightness can be ten times faster than previously established which opens new possibilities for time resolved quantification. The findings stress that the choice of analysis parameters is vital and provide an objective measure of fluorophore eligibility for CoPS. I characterized sixteen organic dyes across the visible spectrum based on their molecular brightness and photostability. This study accomplished the transition of CoPS from a proof of concept technique to a widely applicable quantification method. Experiments demonstrated that CoPS can reveal the label number distribution of fluorescent markers, a prerequisite for quantitative investigations in biology. Moreover, I showed that CoPS offers new perspectives for characterization of photophysical processes in photoluminescent materials