Advancing Single-Molecule Fluorescence Spectroscopy and Super-Resolution Microscopy with Organic Fluorophores

iii-The manifold exciting applications of fluorescence in biophysics, nano-technology or super-resolution microscopy, pose extraordinary and multiple demands on the (single) organic dye molecules employed. A high extinction coefficient together with a high fluorescence quantum yield and water-solubility are nowadays considered as standard properties of suitable organic dyes. However, single-molecule fluorescence resonance energy transfer (single-molecule FRET) and recently introduced super-resolution techniques (termed STORM, PALM, STED, SSIM,...) additionally require a strong resistance against photobleaching and specific functionalities, e.g., stable & non-fluctuating vs. blinking fluorescence intensity, even on the level of single molecules. It is the aim of this work to understand the influence of the specific fluorophore environment, i.e., the composition of the aqueous buffer system needed for the respective (biophysical) application, on the emission properties of organic fluorophores and to utilize this knowledge for the exquisite control of their fluorescence properties. Inducing and quenching radical ion states by photo-induced electron transfer is used to depopulate reactive intermediates such as triplet states to minimize photobleaching. Therefore a buffer system, which contains reducing as well as oxidizing agents (ROXS), is introduced. Its working principle is explained on the basis of thermodynamic considerations of the underlying redox reactions, yielding a comprehensive and unifying picture of blinking and photobleaching of organic fluorophores. In detail, it is demonstrated that ROXS substantially increases the photostability of organic fluorophores from different dye-classes. Simultaneously, blinking is dramatically reduced, enabling fluorescence spectroscopy and imaging applications at higher fluorescence count rates over extended periods of time. In a second step it is demonstrated that a careful selection of fluorophore properties (redox-properties) and environmental conditions (buffer additives) allows the use of ordinary fluorescent dyes as efficient single-molecule switches: Addition or removal of reducing or oxidizing agents switches the fluorescence of several oxazine dyes between stable fluorescent and non-fluorescent dark states. At low oxygen concentrations, the OFF-state – ascribed to a radical anion – is thermally stable with a lifetime in the minute range. The molecular switches show a remarkable reliability with intriguing fatigue resistance even on the single-molecule level with up to 3000 switching cycles. For the first time conventional organic fluorophores were used for super-resolution microscopy using subsequent localization of single blinking molecules (" Blink Microscopy ") under biological relevant conditions, as shown by super-resolution imaging of actin filaments and actin filament bundles in fixed cells with sub-diffraction resolution. Further, " blinking " was used as an observable which sensitively reports …