Developing Switchable and Functional Fluorophores For Multi-Functional Super-Resolution Microscopy

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