PUB - Publikationen an der Universität Bielefeld

Super-resolution fluorescence microscopy (optical nanoscopy) allows us to surpass the optical diffraction limit and observe biological phenomena at the nanoscale using visible light. Different modalities come with their own trade-offs, such as imaging speed vs. achieved resolution or live cell imaging feasibility. In order to achieve axial super-resolution, Total internal reflection fluorescence (TIRF) is often used. Total internal reflection (TIR) of light reflected at the boundary to the sample medium creates an evanescent field in the sample. This sheet-like illumination only reaches a few hundred nanometers into the sample before decaying, thus achieving axial super-resolution. Although it is usually created by using oil immersion lenses, TIR illumination can also be created by coupling light into a high-index nanometer thin layer, called a waveguide. The resulting evanescent fields are independent of the detection optics and can be generated so as to be homogeneous over large scales. Photonic waveguide chips have previously been manufactured using high-index, inorganic materials chemically deposited on silicon substrates. These chips are opaque and costly to produce. This thesis describes the creation of a cost-effective transparent waveguide chip for inverted microscopes and explores its uses in fluctuation-based fluorescence nanoscopy. Secondly, the thesis describes how to exploit the radical transfer between sulfite radicals and glycerol for switching of fluorophores in localization microscopy. The generated radical can be used as a switching agent in imaging buffers for direct Stochastic optical reconstruction microscopy (dSTORM). This novel switching buffer lowers the complexity of dSTORM experiments by being simpler to prepare. In addition dSTORM measurements can be performed earlier after application of the buffer and at lower light intensities, while still achieving high switching speed and low background signal. This switching buffer outperforms ordinary dSTORM buffers with its fast dye switching speeds and high localization densities, being especially useful for samples embedded in hydrogels and high viscosity media. Lastly, the two inventions are combined in order to achieve single molecule localization microscopy on transparent polymer waveguide chips.