Project Summary The overarching goal of the PI’s research program is to decode the intricate biophysical and biochemical functions in diverse molecular processes underlined by the interplay among individual biomolecules and the nanoscopic local environments. To achieve this goal, the proposed research program will focus on the tailor- design of switchable and functional fluorophores and the integration of the probes with multi-dimensional single- molecule imaging (md-SMI) techniques for multi-functional super-resolution microscopy (mf-SRM) and spatially- resolved single-cell multi-omics. Super-resolution optical microscopy has enabled the visualization of subcellular structures and protein molecules with resolution down to the true molecular level (< 5 nm). However, the functional information (i.e., how the biomolecules and the environments interact with each other) within the complex biological system remains elusive. A major bottleneck in decoding the functional information at the single-molecule level is the incompatibility of the existing fluorophores for mf-SRM. Specifically, the (switchable) fluorophores used in existing super-resolution microscopy are predominantly exploited to label and locate the biomolecules and engineered to impose minimal changes responding to the environment thus incompatible in functional imaging. Fluorescent sensors, on the other hand, have been extensively explored to report environmental changes by capturing fluorescent responses (e.g., spectra, lifetime, polarization, intensity) to the environment at the ensemble level. Yet, conventional fluorescent sensors are dim, non-switchable, and lack subcellular molecular specificity, which are key requirements to operate mf-SRM. To address this challenge, therefore, we propose to develop a toolbox of fluorescent sensors as single-molecule functional probes toward mf-SRM. Four research thrusts will be pursued: (1) chromophore engineering to decouple intrinsic fluorescence inhomogeneity at the molecular level from its nanoenvironment factors for hyperplexed mf-SRM strategy for complex molecular interaction imaging related to the chromatin remodeling processes and their coordination with transcription factors, (2) leveraging the development of single-molecule biophysical probes to study the role of the physiochemical functional changes within the higher-order chromatin structure packing/unpacking processes on the epigenetic, (3) developing photon upconversion-based fluorophores for simultaneous near-infrared excitation towards parallel super-resolution interaction and functional imaging. The successful accomplishment and dissemination of the proposed functional fluorophores and enabled mf-SRM imaging capabilities in (a) multi- molecular interaction and (b) mapping the distinct nanoscale biophysical landscapes of subcellular architectures in live cells are anticipated to have an immediate impact on enabling new experimental and computational studies in the fields of chromatin epigenetics and spatially-resolved omics. The newly developed fluorescent probe toolboxes will further promote scientific discoveries and innovations in a wide range of biomedical questions in the fields of epigenetics, chromatin biology and cell biology

PROJECT NARRATIVE This project aims to develop a library of switchable and functional fluorescent probes for multi-functional single-molecule super-resolution imaging of live cells. I will encode distinct single-molecule spectral features of the switchable fluorophores and integrate them with a spectroscopic single-molecule optical imaging platform in the three research thrusts to collectively achieve multi-functional nanoscopic imaging capabilities. This project will allow biomedical researchers, for the first time, to achieve simultaneous interaction imaging of multiple molecules and functional imaging of their surrounding nanoenvironment which fundamentally regulate distinct subcellular processes.