Unravelling chemical and biological processes with advanced probes and enhanced resolution

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Super resolution fluorescence methods based on single molecule localization microscopy (SMLM) are revolutionizing our understanding of biology, chemistry, and physics. Paradoxically, for successful SMLM imaging, high fluorophore density is required for improved resolution, yet only one fluorophore within a diffraction limited spot can be localized at a time. A solution to this paradox has relied in devising fluorophores that cycle between on states and off states. Only a subset of probes is thus recorded and successfully localized at a given time. Images are reconstructed superposing all the localizations. To address the above paradox, we propose innovative ideas to steer photophysical/photochemical pathways in new and existing probes, to ensure controlled cycling and improved super resolution. We also propose developing not just positional beacons (probes that are tagged to and report on the location of substrates of interest) but truly reactive probes. These are probes that activate in response to a chemical cue (e.g. presence of a reactive oxygen species) resulting in "chemical flares". Imaging fluorophore chemo-activation will expand the utility of SMLM to visualize chemical dynamics in 2D and 3D, something we propose to explore in cellular systems. We next propose to use SMLM to build actuatable biocompatible nanostructures based on DNA. These structures will constitute, coupled to SMLM, biophysical tools to explore emerging concepts such as phase segregation in cellular systems (liquid-liquid phase separation and formation of membraneless organelles). Ours is a multi-layered, multifaceted approach articulated along three goals: I. Unravelling fundamental photoprocesses toward innovative fluorescence imaging methodologies. II. Exploiting super resolution imaging and probes to map and decipher the redox chemistry of the cell. III. Providing rules to guide the assembly of next-generation actuatable DNA-based nanomaterials, new tools to image and probe the cell. Our vision is that by judiciously applying chemistry principles, we will contribute transformative advances in fluorescence imaging. In turn, upon exploiting revolutionary fluorescence methodologies, we may unravel chemical processes in, yet, unexplored dimensions. Our mechanistic insights on fluorophore control and photostabilization will translate to unsurpassed resolution in multicolor imaging. Theranostic strategies based on activatable sensitizers will emerge. Our multidisciplinary research approach will provide guiding rules toward constructing complex chemical systems. It will also render unique approaches toward reconciling the redox chemistry with the biology of the cell. The work proposed will translate to a wide range of applications, including nanomaterials, diagnostics and imaging enabling disruptive innovations in biotechnology, materials, and biology to occur. Progress in these areas will positively impact Canada's social and economic wellbeing.