Infoscience

Thesis

Single Molecule Sensing

Living organisms consist of cells, the elementary components of which are proteins providing cellular structures and functionality. Nowadays, proteins of the size of one to a few tens of nanometers can be efficiently monitored by fluorescence microscopy and/or spectroscopy reaching the necessary single molecule sensitivity. However, for a more complete understanding of biological processes, the combined investigation of both cellular structures and function at the single molecule level is essential. This work extends the possibilities of existing single molecule investigation methods. The goal was to conceive a functional super-resolution imaging technique by joining the strong concepts from state-of-the-art microscopy and spectroscopy and was approached in three steps. As a first step, confocal fluorescence correlation spectroscopy (FCS) was applied in nanochannels to create sub-diffraction sampling volumes. The resulting enhanced sensitivity to the surface effects depending on ionic surface layers led to a modified protein diffusion measured with FCS. Triplet state lifetime and population analysis have confirmed the model developed for the protein diffusion as a function of the ionic concentration. In the second step, stochastic optical reconstruction microscopy (STORM) was implemented and enhanced by merging with a recent labeling technique called SNAP-tag. As a result, the labeling technique for bright and switchable organic fluorescent dyes attached by a short linker of only ≈ 2.5 nm to the target molecule enabled a localization precision down to ≈ 10 nm. To meet the stringent photo-physical requirements imposed by STORM, toxic imaging buffers based on thiols have to be used. It was found that these requirements could be relaxed by super-resolution optical fluctuation imaging (SOFI) making a step forward to live-cell super-resolution investigations, which introduced the third step. In the third step, SOFI was characterized providing the basis for combining functional and structural super-resolution imaging techniques. The comparison of STORM and SOFI revealed the strengths of each technique. Under ideal blinking conditions with ultra-stable long-lived dark states, STORM outperforms SOFI. However, if the emitters are not well isolated, SOFI achieves a higher effective resolution than STORM and even without mislocalization artifacts. This led onto a novel imaging technique derived from SOFI: Cumulant microscopy merges structural super-resolution with functional imaging. Simulations and measurements showed that photo-physical parameters can be extracted from the emitters serving as nano-sensors. In conclusion, concepts of FCS have been merged with an improved super-resolution technique leading to a novel functional super-resolution imaging technique, which is compatible with living cells.

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