The aim of the work described in this thesis is to improve the understanding, implementation, and overall capabilities of structured illumination microscopy (SIM). SIM is a superresolution technique that excels in gentle live-cell volumetric imaging tasks. Many modalities of SIM were developed over the last decade that tailored SIM into the versatile and powerful technique that it is today. Nevertheless, the field of SIM continues to evolve and there is plenty of room for novel concepts. Specifically, in this thesis, a generalised framework for a theoretical description of SIM variants is introduced, the constraints of optical components for a flexible SIM system are discussed and the set-up is realised, the important aspect of deconvolution in SIM is highlighted and further developed, and finally novel SIM modalities introduced that improve its time-resolution, gentleness, and volumetric imaging capabilities. Based on the generalised theory, the computational steps for the extraction of superresolution information from SIM raw data are outlined and the essential concept of spatial frequency un-mixing explained for standard SIM as well as for multifocal SIM. Multifocal SIM hereby acts as a parallelised confocal as well as widefield technique and thus serves as link between the two modalities. Using this novel scheme deconvolution methods for SIM are then further developed to allow a holistic reconstruction procedure. Deconvolution is of great important in the SIM reconstruction process, and hence rigorous derivations of advanced deconvolution methods are provided and further developed to enable generalised ‘multi-image’ Richardson-Lucy deconvolution in SIM, called joint Richardson-Lucy deconvolution (jRL). This approach is demonstrated to robustly produce optically sectioned multifocal SIM images and, through the incorporation of a 3D imaging model, also volumetric standard SIM images within the jRL framework. For standard SIM this approach enabled acquisition speed doubling, because the recovery of superresolved images from a reduced number of raw frames through constrained jRL was made possible. The method is validated in silico and in vitro. For the study of yet faster moving samples deconvolution microscopy is found to be the method of choice. To enable optical sectioning, a key feature of SIM, in deconvolution microscopy, a new modality of optical sectioning microscopy is introduced that can be implemented as a single-shot technique. Via polarised excitation and detection in orthogonal directions in conjunction with structured illumination the theoretical framework is rigorously derived and validated.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:744755 |
| Date | January 2018 |
| Creators | Ströhl, Florian |
| Contributors | Kaminski, Clemens F. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/275353 |
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