Advanced Data Processing in Super-resolution Microscopy
von Simon Christoph Stein
Datum der mündl. Prüfung:2017-08-14
Betreuer:Prof. Dr. Jörg Enderlein
Gutachter:Prof. Dr. Jörg Enderlein
Gutachter:Prof. Dr. Holger Stark
EnglischSuper-resolution fluorescence techniques enable the study of structures smaller than the diffraction limit with visible light microscopy. Their introduction and development within the last two decades opened up the possibility to ask entirely new questions in cell biology. The field of superresolution microscopy is still growing rapidly, and many improvements and novel methods have been proposed in recent years. The major part of this thesis revolves around the advancement of Super-resolution Optical Fluctuation Imaging (SOFI), a fairly new technique which enhances the spatial resolution of an image by evaluating the temporal fluctuations of blinking fluorescent emitters. SOFI enables optical sectioning with wide-field microscopes and is compatible with a large range of experimental conditions. The following SOFI-related contributions are presented: First, a comprehensive analysis of the convergence properties of SOFI and its dependence on experimental parameters is shown, including an estimation of the necessary recording time and particle density for a targeted resolution enhancement. Second, we present Fourier SOFI, a new approach to generate super-resolved images on a finer pixel grid than the original camera recording. In contrast to established algorithms relying on spatial cross-cumulants, this method is practically free of artifacts and does not require any post-processing corrections. Next, an algorithm is laid out that corrects for the contributions of noise in zero-time-lag SOFI images. This extends the applicability of auto-cumulant SOFI – which correlates values only in time, not in space – to recordings where the time scale of photoblinking is on the same order as the exposure time. We also show how the lateral microscope Point Spread Function (PSF) can be estimated from SOFI data of thin samples. If multiple focal planes are imaged at the same time, the full three-dimensional PSF can be recovered. The fifth contribution revolves around Fourier Preweighting, a method that increases the resolution of SOFI images by pre-processing the original data. We show that this outperforms current techniques and surprisingly also improves the density dependence of SOFI. A related algorithm is presented which automatically matches the degree of resolution enhancement to the data quality, avoiding artifacts. The last SOFI contribution demonstrates its applicability to cells labeled with carbon nanodots, a fairly new cost-effective and bio-compatible class of fluorophores, and shows that their blinking behavior is qualitatively similar to that of fluorescing semiconductor crystals (quantum dots). Unrelated to SOFI, we show how single Atto647N molecules can be localized with sub-nanometer precision by exploiting the increased photon yield of samples at liquid nitrogen temperature. We propose a method to resolve spectrally identical, non-blinking fluorophores spaced only few nanometers apart with a special cryo-fluorescence setup and validate our concept with simulations. We also developed TrackNTrace, an open-source MATLAB framework to support the development of fluorescence imaging applications. Its design is focused on easy extensibility through plugins, simplicity of coding, and rich visual feedback. We demonstrate competitive performance and execution speed in Single-Molecule Localization Microscopy applications compared to established software and include many state-of-the-art algorithms out-of-the-box. Finally, we present two projects reliant on single-molecule imaging: First, we developed a model for the intensity distribution of fluorescent molecules imaged while flowing through a nanochannel and verified it experimentally. This can be used to extract the ratios of differently labeled species from a measurement of their mixture. In the second project, we demonstrate the first simultaneous measurement of the excitation and emission dipole axes of single molecules.
Keywords: Fluorescence Microscopy; Super-resolution; Super-resolution Optical Fluctuation Imaging; SOFI; Cryo Fluorescence Microscopy; TrackNTrace; Single Molecule