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Applying single-molecule localisation microscopy to achieve virtual optical sectioning and study T-cell activation 10.17863/CAM.16306 Palayret, Matthieu Grégoire Simon Single-molecule localisation microscopy (SMLM) allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. As a single-molecule technique, it has also introduced a new quantitative approach to fluorescence microscopy. In the Part A of this thesis, the design and building of three SMLM instruments, the implementation of a custom-developed image analysis package and the characterisation of the photo-physical properties of the photo-activable fluorescent protein used in this thesis (mEos), are discussed. Then, a new post-processing method for SMLM analysis is characterised: axial optical sectioning of SMLM images is demonstrated by thresholding fitted localisations using their fitted width and amplitude to reject fluorophores that emit from above or below a virtual ‘light-sheet’, a thin volume centred on the focal plane of the microscope. This method provides qualitative and quantitative improvements to SMLM. In the Part B of this thesis, SMLM is applied to study T cell activation. Although the T cell receptor plays a key role in immunity, its stoichiometry in the membrane of resting T cells is still a matter of debate. Here, single-molecule counting methods are implemented to compare the stoichiometry of TCRs fused with mEos2 in resting T cells to monomeric and dimeric controls. However, because of the stochasticity of mEos2 photo-physics, results are inconclusive and new counting techniques based on structural imaging are discussed. In addition to TCR triggering, T cells require the co-stimulatory triggering of the CD28 transmembrane receptor to become fully activated. However, some immobilised anti-CD28 antibodies, referred to as super-agonists (SA), can directly activate T cells without triggering the TCR. In this thesis, single-molecule tracking techniques are used to investigate the molecular mechanism of CD28 super-agonism in live T cells. The results indicate that the diffusion of CD28 is slowed by SA binding. This effect is further discussed in light of the kinetic-segregation model proposed for TCR triggering. Quantitative SMLM as implemented and further developed in this work offers new tools to investigate the molecular mechanisms initiating T cell activation, ultimately facilitating the discovery of novel approaches to target these pathways for therapeutic purposes. University of Cambridge 2015-10-06 Thesis Doctoral Doctor of Philosophy (PhD) en This work was supported by the Wellcome Trust [studentship number 093756/B/10/Z]. https://www.repository.cam.ac.uk/handle/1810/252696 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/acf087e8-ea03-41f0-9641-e1f89313efb4/download dd545931f10f1dfdd1039f529a25cc30 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/9881d771-2789-4d42-8d7d-8339d33d759a/download 5657bfe93973a2cde74f1c00cfd43399 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b7a4e4fe-9e54-4da5-986e-5e258397fed5/download db55dfe2e15f9ed86356ce91665edd1c https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b7c98974-d7b6-40fa-99b6-3de71e45494d/download d5c7604fe919fb73790f3d9fdc023534 https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cd1e45b4-af76-4166-865c-d05318309be9/download c74eeb083cd7198b3eb5de1b5deb482a https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/0f0041f1-c90c-4852-8dc0-bb22a072b35a/download b46c912889e1d8a940bc42f7385365ad https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/07708f3d-ae82-4415-832b-237635f911fc/download 835269bda140c10400fe0606a14c3d21 T-cell receptor kinetic-segregation CD28 super-agonist single-molecule localisation microscopy super-resolution fluorescence microscopy virtual 'light-sheet' optical sectioning