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Abstract

Fluorescence microscopy techniques have significantly advanced our understanding of biological systems, enabling the investigation of numerous cellular processes with high spatiotemporal resolution. Light as a non-invasive stimulus enhances resolution and fosters interest in innovative photoswitchable systems for modern microscopy techniques such as super-resolution microscopy. This technique requires specific fluorophores that can switch between fluorescent and non-fluorescent states. In the past decade, significant efforts have focused on developing light-responsive fluorescent proteins and small-molecule fluorophores. While fluorescent proteins are genetically encodable, they often exhibit low brightness and photostability, particularly in the far-red region. Conversely, synthetic small-molecule fluorophores generally show superior optical properties but their applications in living cells are limited, due to a lack of reversible and controllable systems. In this thesis, I present novel approaches for the spatial and temporal control of fluorescence by designing systems that utilize bright, fluorogenic rhodamine derivatives in conjunction with the self-labelling protein HaloTag. By incorporating a light-responsive moiety into this hybrid scaffold, I demonstrate the potential for reversible modulation of the fluorophore's emission. I explored two strategies: one employing small-molecule photoswitches (azobenzenes) and the other utilizing genetically encoded photoswitchable proteins (AsLOV2). Although I achieved proof-of-principle for reversible fluorescence modulation upon illumination, all azobenzene-rhodamine dyads exhibited strong fluorescence quenching, which limited their utility in fluorescence microscopy applications. A more promising approach emerged by incorporating the AsLOV2 domain into HaloTag. I engineered various photoswitchable proteins and identified candidates that exhibited reversible far-red fluorescence turn-on upon illumination. These engineered proteins were successfully demonstrated in mammalian cells across various targets, achieving a several-fold increase in fluorescence upon illumination. These results suggest that this system holds promise for super-resolution imaging by allowing fine control over the blinking behavior of self-blinking dyes. Additionally, I explored an application of photoresponsive systems using a recently introduced photoclick reaction to enable highly multiplexed optical labelling in living cells with high spatiotemporal control. This approach facilitates on-demand marking of specific cells or subcellular features for real-time tracking and analysis of their dynamics. Currently, this is achievable using photoactivatable synthetic dyes or proteins, however, such methods are limited to single-color experiments. To address these limitations, I developed photoclickable ligands for HaloTag and SNAP-tag that can covalently bind to clickable fluorophores upon illumination. These newly developed probes show high performance in vitro, in E. coli, and on the surface of living mammalian cells, demonstrating effective light-controlled fluorescence labelling. Keywords: fluorescence microscopy, photoswitch, fluorophore, rhodamine, HaloTag