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978-3-8439-3212-7, Reihe Biophysik
Jochen Michael Reichel
Design, implementation, evaluation and application of a dual color 3D single molecule localization microscope
174 Seiten, Dissertation Universität Ulm (2016), Softcover, B5
Super-resolution far-field optical microscopy, or nanoscopy, has emerged in recent years and has revolutionized the field of fluorescence microscopy in biophysical, biological and biomedical research. Among the many different nanoscopy techniques single molecule localization microscopy (SMLM) has shown to provide the best routine resolution enhancement of optical far-field microscopy so far. This is achieved by replacing the point spread function (PSF) of the single fluorescing molecules by a point at their individual central positions that can be determined with high accuracy and the collection of points eventually constitutes the super-resolved image. The technique is mainly limited by the maximum number of photons that can be collected from the labeling sites above background noise. Also the labeling density strongly influences the achievable resolution of the final image
In this thesis, a custom-built dual color 3D SMLM microscope is presented and its performance is probed in detail. Also the extension to a dual objective setup for enhanced photon collection efficiency is described. Localization precisions (FWHM) of ~22nm in the lateral direction and of ~72nm in the axial direction are achieved with the single objective configuration. The position of the sample could be stabilized in the axial direction with a custom-built focus lock system to 1.2nm (SD) over the time span of 10 minutes. For the lateral direction however residual drifts of 10-200nm within 20 minutes are observed, giving rise to the necessity of post-process drift correction.
This post-processing including analysis and reconstruction of the localization data as well as background correction and drift correction is executed with a custom-written software based on center of mass calculation or 2D integrated Gaussian fitting. This software termed FIRESTORM (fluorescent spot identification and reconstruction of STORM data) is described and tested in detail with the help of simulated and real data.
The superior resolution and biological applicability of our SMLM microscope is finally demonstrated with the investigation of the exact position of the protein fused in sarcoma (FUS) in synapses of cultured rodent embryonic hippocampal neurons. The protein FUS is found to locate on the presynaptic side. In another study the change of morphology of the microtubule (MT) network in HeLa cells upon overexpression of the RBP-J interacting and tubulin associated protein (RITA) is investigated.