Exploiting TICT to Enrich Fluorophore Information for Single-molecule Diagnostics

With the support of the Chemical Mechanism, Function, and Properties Program of the Division of Chemistry, Professors Kristin Wustholz and Elizabeth Harbron of the Department of Chemistry at the College of William and Mary are developing new fluorescent dyes with unique properties for single molecule spectroscopy (SMS) applications. SMS is renowned for its ability to visualize complex materials and biological structures that are important in technology and medicine. Current SMS studies focus on brightly fluorescent materials that emit light continuously until they fail and become dark. However, some fluorescent dyes can switch between bright, dim, and dark states, with these fluctuations containing important information about the molecule, its location, and its environment. By combining expertise in chemical synthesis, SMS, and advanced statistical analysis, the team of Wustholz and Harbron is creating new dyes engineered to produce dim states and deploying them to reveal otherwise inaccessible structural and dynamic information at the single-molecule level. Students supported by this project gain a range of technical expertise. Considerable effort has been devoted to designing fluorophores that exhibit ideal fluorescence properties such as high quantum yield, which requires minimizing nonradiative deactivation pathways. Minimizing twisted intramolecular charge transfer (TICT), where an internal donor-acceptor charge transfer occurs upon molecular twisting, has improved quantum yields in rhodamines and other common dyes. Conversely, maximizing TICT creates non-emissive dyes that can be useful as fluorogenic probes. While dye design efforts in these highly fluorescent and fluorogenic regimes have led to significant advances in nanoscale imaging, they also discard useful information. This proposal harnesses this untapped wealth of information in an intermediate “diagnostic” regime for single molecule imaging. The team is making a series of TICT-active fluorophores, determining how TICT manifests in single molecule intensity fluctuations (blinking dynamics), and establishing quantitative metrics that contain diagnostic information. Additionally, by deploying TICT dyes to image biological systems, this project is examining one-color multiplexed imaging that reports more fully on environmental spatial and temporal heterogeneities. Collectively, this project generates new probe design strategies and advances fluorescence studies of complex materials and biological systems. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.