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Single Molecule Studies

Fluorescent techniques nowadays offer extremely high sensitivities and thus principally allow to detect and to examine individual molecules. Reaching such high sensitivities is still challenging because intrinsically, signals from single molecules are relatively weak and background contributions have to be suppressed sufficiently. However, a wealth of information can be gained from these experiments which are not accessible in ensemble measurements. The most prominent advantage of Single Molecule (SM) studies is avoiding ensemble averaging. Thus, individual molecular properties and their evolution over time are revealed and quantitative measures are directly provided. Especially when different molecular states are involved, SM experiments give insight in state distributions and populations which often average out in ensemble measurements.

Practically, low concentrations and small observation volumes are necessary to study molecules individually. In the diffraction limited effective volume of a confocal microscope, bursts of freely diffusing single molecules usually can be measured for concentrations in the picomolar to nanomolar range. Higher concentrations are accessible working with micro and nano containers such as vesicles, polymerosomes, nanodiscs or Zero-Mode-Waveguides (ZMWs). Alternatively, imaging individual molecules immobilized on surfaces with low density allows measurements for more extended time periods, typically in the range of seconds to minutes. Both confocal microscopy and wide-field (in particular in TIRF (Total Internal Reflection Fluorescence) excitation mode) microscopy are well suited to study surface-bound molecules.

We study conformational dynamics of proteins upon co-translational and post-translational folding (and unfolding). Additionally, we investigate structural fluctuations in the native state and upon substrate binding. Therefore, SM fluorescence experiments are carried out in solution as well as on surfaces using time-resolved confocal microscopy, wide-field fluorescence and TIRF microscopy.

Contact: Tina Züchner