{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/252696"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/252696","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Applying single-molecule localisation microscopy to achieve virtual optical sectioning and study T-cell activation","abstract":"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.","abstract_html":"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.","abstract_has_math":false,"creators":["Palayret, Matthieu Grégoire Simon"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2015,"date_issued":"2015-10-06","date_published":"2015-10-06","updated_at":"2026-07-22T22:24:32Z","subjects":["T-cell receptor","kinetic-segregation","CD28 super-agonist","single-molecule localisation microscopy","super-resolution fluorescence microscopy","virtual 'light-sheet'","optical sectioning"],"languages":["en"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/07708f3d-ae82-4415-832b-237635f911fc/download"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.16306","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.sponsor","label":"Sponsor","values":["This work was supported by the Wellcome Trust [studentship number 093756/B/10/Z]."]},{"key":"dc:creator","label":"Author","values":["Palayret, Matthieu Grégoire Simon"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2015-10-06"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/252696"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["T-cell receptor","kinetic-segregation","CD28 super-agonist","single-molecule localisation microscopy","super-resolution fluorescence microscopy","virtual 'light-sheet'","optical sectioning"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/07708f3d-ae82-4415-832b-237635f911fc/download"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["10.17863/CAM.16306"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/acf087e8-ea03-41f0-9641-e1f89313efb4/download","https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/9881d771-2789-4d42-8d7d-8339d33d759a/download","https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b7a4e4fe-9e54-4da5-986e-5e258397fed5/download","https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/b7c98974-d7b6-40fa-99b6-3de71e45494d/download","https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/cd1e45b4-af76-4166-865c-d05318309be9/download","https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/0f0041f1-c90c-4852-8dc0-bb22a072b35a/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Single-molecule localisation microscopy (SMLM) allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. 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