Photoswitchable Dyes for Super-Resolution Microscopy - Super-resolution microscopy, i.e., nanoscopy, breaks away from the ~200 nm resolution restriction of optical microscopy, known as the Abbe diffraction limit, opening the way for the optical imaging of small molecules that are at the core of biological processes. In principle, nanoscopy techniques can result in real-time, 3D, single molecule imagining in live specimens. To this end, molecules whose emission can be switched ON and OFF are required, and while there has been much progress in this area, the development of fluorescent molecules with appropriate properties (e.g., high ON/OFF contrast, photo-stability, fatigue resistance, red-shifted activation/emission, fast kinetics, biocompatibility, etc.) has proved to be extremely challenging. For example, while there are reversible photoswitchable proteins, their emission wavelengths and quantum yields are limited and/or low. On the other hand, reversible organic dyes might be brighter but suffer from poor biocompatibility, small contrast ratios, photobleaching and so on. Addressing these issues will be critical for nanoscopy to realize its full potential - the molecular level visualization of biological processes as they occur. My group has developed a new family of bistable hydrazone-based switches whose emission can be toggled ON and OFF using different wavelengths of light in organic and aqueous solutions (including serum), the solid-state and even in HeLa cells. The switches have very good absorption coefficients and emission quantum yields, and hence brightness. The combination of these properties in a structurally simple platform that can be easily synthesized and derivatized is unique and highly desirable, making this family of switchable fluorophores highly competitive with protein or organic dye-based switchable fluorophores. The overarching goal of this MIRA proposal is to transform these switchable hydrazones into an array of multifunctional, bright, tunable, water-soluble, biocompatible, and conjugatable dyes with broad potential utility in nanoscopy. As part of the grant, we will apply a multipronged approach to enhance the photophysical properties of the dyes through hypothesis-driven structure-property analyses, which will be aided and guided by DFT calculations, combined with spectroscopic characterization including bio-imaging. Ultimately, and through feedback loops that will guide us in improving our design principles and hypotheses, we will gain the fundamental insights needed to transform the switchable hydrazones into probes that can be used in multiplexed live-cell imaging using nanoscopy. Such an accomplishment will in turn expand the toolbox available to practitioners, while enhancing the window of application opportunities available to them. As part of our efforts, we will also design switchable fluorophores that can selectively bind to soluble amyloid (A) oligomers, so they can be imaged using nanoscopy. These studies will provide insights and high-resolution images of the oligomer dynamics, structures and interactions, which are crucial for understanding their pathology and, consequently, for advancing the development of therapeutics and early diagnostics for neurodegenerative diseases.
