Development of Photoactivatable Fluorophores to Explore Physicochemical Environments in Live Cells and Access Long-Term Single-Molecule Imaging

Abstract

Fluorescence microscopy is an invaluable tool for biological research, used routinely due to its high sensitivity, low invasiveness, and the rich phenotypic information it provides. The variety of available fluorescent probes allows for acquiring diverse information about the sample, from analyte concentrations, through properties of the microenvironment, to biological activity. An important constraint of fluorescence microscopy is its resolution, which is limited by the diffraction of light, making these techniques unable to detect biologically relevant events at the scale of macromolecules. Super-resolution techniques like single-molecule localization microscopy (SMLM) overcome the limit of diffraction and offer an insight into these microscale processes with a resolution in the range of tens of nanometers. SMLM techniques rely on photochemically active fluorophores with an emissive and a non-emissive state and the possibility of switching between them. There is a need for probes with minimal phototoxicity from switching as this poses a limit on the duration of SMLM experiments. Photoactivatable fluxional fluorophores offer a solution by using irradiation pulses only to promote precursor molecules to a dynamic equilibrium, where thermal rearrangements facilitate the switching of molecules between a fluorescent and non-fluorescent form. This strategy is advantageous because it allows for photo-control of the number of emitters with reduced phototoxicity relative to other methods that use high frequency, short-wavelength irradiation for this purpose. Currently, the only examples of photoactivatable fluxional fluorophores are rhodamine acyl hydrazone derivatives that have their specific application in labeling acidic vesicles. In this thesis, we report our efforts to generalize photoactivatable fluxional fluorophores with HaloTag binding rhodamine and silicon rhodamine molecules. The site of HaloTag functionalization was optimized, and a set of probes with different photoisomerizing functional groups were synthesized and evaluated in microscopy experiments. Photoactivatable fluxional behavior was reproduced in non-acidic environments with in the HaloTag bound state of the developed dyes. Not only the low invasiveness of conventional fluorescence techniques is compromised for the higher resolution in SMLM methods, but also the repertoire of functional sensors needs improvement. Polarity-sensitive fluorophores contribute a large share to the versatility of conventional fluorescent imaging methods, but polarity-sensitive probes so far are not available in SMLM imaging techniques. In this thesis, we report elucidation of the polarity-sensitive photoactivation mechanism of diazoindanone-masked silicon rhodamines. We developed a fatty acid probe to label lipid droplets, verified it in model systems and subsequently used it for labeling the periphery of droplets in live cells for SMLM imaging with reduced background. The photoactivatable property of the probe was used to observe lipid transport between lipid droplets and the endoplasmic reticulum. Furthermore, the polarity-sensitive photochemistry of diazoindanone masked silicon rhodamines was also for polarity sensing to detect liquid-liquid phase separation. In this context, we applied a peptide-targeted probe to investigate the microenvironment of chromatin in live cells. With single-molecule tracking, we could additionally employ our probe to infer viscosity information about the heterochromatin. In these projects, we developed probes to address some of the limitations of SMLM techniques. We could reduce phototoxicity of the method using photoactivatable fluxional fluorophores and were able to add polarity-sensitive probes to the range of available functional reporters.