Super-resolution fluorescence microscopy, which allows the resolution of features below the diffraction limit of light, has transformed our ability to investigate cellular processes on the nanoscale. This thesis describes the synthesis, characterisation and testing of photoswitchable fluorescent dyes for application in RESOLFT super-resolution microscopy.

Chapter 1 introduces super-resolution fluorescence microscopy and the many different techniques for resolving below the diffraction limit. It goes on to explore the differences in the chemical tools (fluorescent dyes) required for each of these techniques. Finally, it focuses on the requirements for photoswitchable dyads for RESOLFT in particular, and explores some molecular design principles.

Chapter 2 describes a dyad which combines environmental polarity sensing with RESOLFT super- resolution imaging. Polarity sensing below the diffraction limit is demonstrated in synthetic vesicles, a first for RESOLFT microscopy.

Chapter 3 reports the synthesis and characterisation of a new FRET quenching photoswitchable dyad based on a diazocine photoswitch, in an attempt to improve the fatigue of previous FRET dyad designs. The photophysical properties of this dyad are thoroughly characterised, and a more complete model for photoswitching is developed based off its unexpected behaviour.

Chapter 4 explores PET quenching as a mechanism for photoswitchable dyads, exploiting distance changes between a quencher and a dye to modulate electron transfer rates in competition with fluorescence. The synthesised dyads show important proof of concept results, which inform the development of more rigid and close-packed designs towards this goal.