Single-molecule microscopy study of nano-systems

In this work, techniques were developed and used to study the properties of molecules on a single-molecule level. Single-molecule techniques have the major advantage, that in contrast to ensemble measurements, they allow a detailed insight on the distribution and dynamics of single molecules without averaging over subpopulations. The use of Total Internal Reflection Fluorescence Microscopy (TIRFM) in combination with single-pair Förster Resonance Energy Transfer (spFRET) and Alternating Laser Excitation (ALEX) allows the identification of molecular-states by making quantitative measurements of distances in the Ångström range. The development of highly sensitive photon detectors and the use of versatile labeling techniques with photostable (synthetic or genetically-encoded) fluorophores, extended the application of TIRF microscopy to in vitro and live-cell experiments. Despite reducing the complexity of biological systems down to the single-molecule level, functions of individual molecules and interactions between them can be very sophisticated and challenging to analyze. Using information theory based methods, e. g. HMM, the dynamics extracted from single-molecule data was used to illuminate protein interactions and functions.