Single Marker Switching Nanoscopy with 4Pi Detection: Superior Nanometric 3D Resolution

The spatial resolution of light microscopy was limited by diffraction for a long time. Switching markers between a dark and a bright state was the key element to overcome this limitation. In single marker switching (SMS) microscopy, switching and subsequent localization of single markers enables the creation of two-dimensional superresolution images. The approaches to extend SMS microscopy to the third dimension were hitherto hampered by a substantially anisotropic resolution or limited to samples thinner than half the marker fluorescence emission wavelength. Still, a light microscope allowing three-dimensional subdiffraction imaging of thick samples with a homogeneous and isotropic resolution combined with the ability to differentiate between different types of markers is highly desirable. This thesis presents a nanoscope which combines the SMS concept with the 4Pi-technique to address these important issues. The spherical wavefronts generated by a single fluorescence emitter are collected by two opposing objective lenses of high numerical aperture, brought to interfere and focused onto an area detector. By evaluating higher order moments of the detected spots, single markers can be simultaneously localized within samples of theoretically unlimited thickness with an unsurpassed accuracy. 3D localization accuracies of better than 10 nm within a layer of at least 1 micrometer thickness are shown. Importantly, this 1 micrometer sheet can be placed at any depth within the sample. Further, by splitting the fluorescence into orthogonal polarization states, the 4Pi-SMS setup facilitates the 3D nanoscopy of multiple marker species. The biological applicability of the presented method is proven for different biological samples and for distinct types of markers. Offering a unique combination of multicolor recording, nanoscale resolution, and extended axial depth, this work substantially advances the non-invasive 3D imaging of cells and other transparent materials