Kompaktní Mikroskopie se Strukturním Osvětlením a Hodnocení Výkonu v Superrezolučním Zobrazování ŽIvých Buněk

• Main Author: Pospíšil, Jakub
• Format: Dissertation
• Language: Czech
• Published: ProQuest Dissertations & Theses 01-01-2021
• Subjects: Astronomy; Biology; Collaboration; Digital imaging; Lasers; Localization; Microelectromechanical systems; Microscopes; Microscopy; Optics; Power; Pulmonary arteries; Stars & galaxies
• ISBN: 9798802730119

Structured illumination microscopy (SIM) is one of the most common means of achieving a spatial resolution beyond the diffraction limit in fluorescence microscopy. The superresolution SIM doubles the spatial resolution of optical microscopy, which is sufficient to visualize cellular organelles and their substructures at _100 nm. Current commercially available SR-SIM microscopes are often unaffordable to many biological laboratories and are relatively bulky systems covering more than one optical table. The high demand for inexpensive compact SR-SIM microscopes leads to the development of the custom-built systems using alternative ways of generating the striped illumination pattern essential for the SIM reconstruction, e.g., using a digital micromirror device (DMD), a ferroelectric liquid-crystal-on-silicon (FLCOS) microdisplay, or fiber optics. This thesis details development of a novel custom-built structured illumination microscopy system based on all-fiber optic components (fiberSIM). The proposed SIM system provides promising super-resolution results confirming the concept and indicates that fiberSIM could become the basis of a versatile “Plug&Play” illumination module with the capability of turning any conventional microscope into the SR-SIM microscope. Furthermore, five freely available SIM datasets with complete documentation are introduced. This SIM data provides a benchmark essential for the further development of reconstruction algorithms or analysis tools in SR-SIM microscopy. The final step in the development of a custom-built SR microscope is the quantitative evaluation of the resolution gain. In this thesis, a recently developed resolution estimation technique based on circular average power spectral density (PSDca) analysis is introduced. The proposed method allows to determine the resolution limit from a single image, which is suitable for the analysis of video sequences of a living cell. Furthermore, the most common conventional resolution assessment techniques that are able to measure the actual resolution limits of a microscope are discussed. The combination of a compact low-cost SIM illumination unit with open-source reconstruction and analysis algorithms makes SR-SIM available to any biological laboratory.