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Vortex Light Field Microscopy for 3D Spectral Single-Molecule Imaging https://doi.org/10.17863/CAM.120397 Zhang, Boya Robinson, Hugh Singe-molecule localization microscopy (SMLM) is a powerful imaging technique that surpasses the diffraction limit and significantly improves spatial resolution over conventional light microscopy, allowing cellular structures and dynamic processes to be visualized at nanoscale. To solve the inherent three-dimensional (3D) architecture of biological structures, various 3D SMLM methods have been developed to extend axial range and to improve axial resolution. Combining 3D with multicolor SMLM methods enables multiple fluorescent probes to be imaged in the same volume, offering opportunities to study the spatial relations and interactions between different species. However, there are only a few methods capable of 3D multicolor singe-molecule imaging, most of which either achieve continuous wavelength detection with a shallow axial range, or extended depth at several discrete wavelengths. Therefore, to address the need of a method for continuous spectral measurement with large axial range, we developed vortex light field microscopy (VLFM), a novel method for simultaneous 3D spectral single-molecule imaging. This thesis outlines the development of VLFM. The working principle of VLFM is f irstly introduced, as well as related theoretical background for disparity calculation and light propagation. Proof-of-concept experiments are performed with a spatial light modulator to prove the concept of VLFM. Then a detailed guide for the optical design is provided, as well as fabrication notes and the final experimental implementation. The latter chapters involve the single molecule applications of VLFM. A reconstruction method is tailored for VLFM, and system characterization is conducted to assess the PSF compactness and localization precision of VLFM.Then VLFM’scapability is experimentally demonstrated by 4-color single particle tracking and multicolor dSTORM imaging in fixed COS-7 cells. The application of VLFM is further extended to the field of diffraction-limited imaging. By combining the previously designed optics with Richardson-Lucy deconvolution algorithm, VLFM is potentially a useful tool for fast multicolor volumetric imaging, which is demonstrated by proof-of-concept simulations. Overall, this thesis contributes a novel 3D multicolor imaging method that pushes the boundaries of current single-molecule imaging techniques, enabling investigations into multiple complex biological structures and interactions at the nanoscale. University of Cambridge 2024-12-25 Thesis Doctoral Doctor of Philosophy (PhD) https://www.repository.cam.ac.uk/handle/1810/387728 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ab3a811a-36f8-4d19-a1cf-c79d28bc8999/download ed37e32ad62ef410ac3564ea3e942476 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/165f0dff-8966-4002-a665-92151c50ea78/download 87eda9de84448d1f82354d60eee3eb5f http://purl.org/NET/rdflicense/allrightsreserved Super-resolution microscopy Single-molecule imaging