Next-generation fluorophores for single-molecule and super-resolution fluorescence microscopy
The development of single-molecule and super-resolution fluorescence techniques has revolutionised biological imaging. Nano-scale cellular structures and heterogeneous dynamic processes are now able to be visualised with unprecedented resolution in both time and space. The achievable localisation precision and therefore the resolution is fundamentally limited by the number of photons a single-fluorophore can emit. The ideal super-resolution dye would emit a large number of photons over a short period of time. On the contrary, an optimal single-molecule tracking probe would be highly photostable and undergo no transient dark-state transitions. Single-molecule instrument development is beginning to reach technological saturation and as the frontiers of bioimaging expand, exorbitant demands are placed on the gamut of available probes that often cannot be met. Thus, the next key challenge in the field is the development of the better fluorophores that underlie these techniques; this includes both the synthesis of new chemical derivatives and alternative novel strategies to augment existing technologies. The results of this thesis are divided into two distinct parts; Project One details the development of new synthetic fluorescent probes for the study of amyloid protein aggregates implicated in neurodegenerative diseases. This includes a study of the photophysical and binding properties of a novel fluorophore library based on the amyloid dye Thioflavin-T. Following on from this, is the presentation of novel bifunctional dyes capable of simultaneously identifying hydrogen peroxide and amyloid aggregates by combining existing tools for the independent detection of these species. The sensing capabilities of these dyes are explored at the bulk and single-molecule levels. Project Two describes a new photo-modulatable fluorescent-protein fusion construct that can undergo Förster resonance energy transfer (FRET) to an organic dye molecule. This FRET cassette is comprised of a photoconvertible fluorescent protein donor, mEos3.2 and acceptor fluorophore, JF646. This strategy imparts a strong photostabilising effect on the fluorescent protein and a resistance to photobleaching. The functionality of this approach is demonstrated with in vitro single-molecule fluorescence studies and its biological applicability shown by tracking single proteins in the nuclei of live embryonic stem cells. Furthermore, initial characterisations of the excited state dynamics in effect are presented through the systematic modification of parameters.