Proteins in our body are usually folded to carry out their physiological functions. However, they can become misfolded and aggregated throughout the lifetime of a cell. These proteins can be degraded or cytotoxic, the latter of which leads to diseases such as neurodegeneration and cancers. Unfortunately, the compositions of native protein aggregates are still not fully known. Mapping them could help us understand the diseases they cause, and provide the basis for early diagnosis and monitoring therapy.

Since protein aggregates are heterogeneous in sizes and structures, single-molecule techniques are required to unravel their compositions. Super-resolution microscopy is particularly useful, as these aggregates are smaller than the diffraction limit of light. Compared to conventional light microscopy, the fluorescence of the nearby molecules in super-resolution microscopy fluoresces at different times. With this, even though the fluorescence of the single fluorophore is still blurred by the diffraction limit (~200 nm), the fluorophore is inferred as in the centre of the blurred spot. Thus, the resolution in super-resolution microscopy can reach down to sub 20 nm. This allows us to super-resolve the protein aggregates and may therefore help us decipher the mysteries of disordered and aggregated proteins. However, applying super-resolution microscopy to these complexes is a challenge. Although there are already a range of developed methods, there are still important limitations.

In this thesis, I present a series of advances in single-molecule techniques for mapping protein aggregates. Firstly, an advanced illumination module for super-resolution microscopy to facilitate accurate measurements was implemented and characterised. Functionalisation of antibodies, which are commonly used to map native protein aggregates in super-resolution microscopy, was then optimised. Next, single-molecule pull-down assays were investigated with the aim of capturing and imaging protein aggregates with higher selectivity, sensitivity, and speed on glass coverslips for super-resolution microscopy. Meanwhile, two classes of DNA-small molecule conjugates, with PET-ligand analogues and aggregate-targeting peptides, were synthesised and tested. These novel probes can potentially selectively target protein aggregates in super-resolution microscopy. Finally, a newly developed, state-of-the-art microscope for super-resolution microscopy, NanoPro, is presented. The theme that unites the presented work is improving single-molecule techniques for mapping protein aggregates.