Applying single-molecule localisation microscopy to achieve virtual optical sectioning and study T-cell activation
Authors
Palayret, Matthieu Grégoire Simon
Advisors
Klenerman, David
Date
2015-10-06Awarding Institution
University of Cambridge
Author Affiliation
Department of Chemistry
Qualification
Doctor of Philosophy (PhD)
Language
English
Type
Thesis
Metadata
Show full item recordCitation
Palayret, M. G. S. (2015). Applying single-molecule localisation microscopy to achieve virtual optical sectioning and study T-cell activation (Doctoral thesis). https://doi.org/10.17863/CAM.16306
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.
Keywords
T-cell receptor, kinetic-segregation, CD28 super-agonist, single-molecule localisation microscopy, super-resolution fluorescence microscopy, virtual 'light-sheet', optical sectioning
Sponsorship
This work was supported by the Wellcome Trust [studentship number 093756/B/10/Z].
Rights
Attribution 2.0 UK: England & Wales
Licence URL: http://creativecommons.org/licenses/by/2.0/uk/
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