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.