Yang Zhang

The goal of this project is to generate a library of cell-permeable and bioconjugatable synthetic dyes with photoactivatable fluorescence and resolved single-molecule emission spectra. Their design is based on a photochemical mechanism to activate borondipyrromethene (BODIPY) fluorescence with green light developed in our prior funding cycle. Their realization demands the synthesis, structural characterization, ensemble/single-molecule spectroscopic analysis and imaging of four series of photoactivatable BODIPYs, differing in the ring-fusion pattern of their polycyclic chromophoric scaffold and/or nature of their substituents, in two parallel project components. One is aimed at resolving the spectral position and/or band shape of the photoactivated fluorescence to differentiate the single-molecule emission spectra sufficiently for structural discrimination with statistical confidence. The other is aimed at introducing hydrophilic groups and targeting ligands on the chromophoric platform to label selectively and track simultaneously intracellular proteins. The overall outcome will be the engineering of a unique combination of structural, photochemical and photophysical properties into innovative molecular constructs to enable the unprecedented high-throughput multiplexed single-molecule tracking of proteins in live cells.

The objective of the proposal is to develop a palette of synthetic photoactivatable fluorophores (PAFs) for live-cell multiplexed spectroscopic single-molecule tracking of multiple (???4) biomolecules simultaneously with 10-nm spatial precision and 10-ms temporal resolution. In particular, the specific aims of this proposal are (1) to synthesize visible-light activatable and bioconjugatable PAFs for spt-PALM (referred to single particle tracking-Phoactivated localization microscopy) with minimal photocytotoxicity and (2) to synthesize PAFs with resolved emission peaks in the red spectral region for simultaneously multiplexed spt-PALM. The photogenerated species of the photoactivatable probes with distinct yet partially-resolved emission spectra will be concurrently localized and distinguished using spectroscopic single-molecule localization microscopy (sSMLM) to achieve multiplexed spt-PALM, thus enabling simultaneous multi-color super-resolution imaging in live cells. The grantee shall perform the single-molecule spectroscopic characterization of the resulting switchable probes after available and super-resolution imaging experiments in live cells. The specific goals for the collaborative research activity at NCSU are (1) the high-throughput measurement of single-molecule fluorescence spectral heterogeneity of the photogenerated species to guide the synthesis of a quartet of photoactivatable BODIPY-oxazine dyads that can be unambiguously distinguished; (2) the acquisition of single-color spt-PALM in live cells of specific subcellular organelles labeled with the developed switchable probes and activate by green light; (3) the development of imaging method to simultaneously track 4 photoactivated BODIPY chromophores and imaging analytics; (4) perform parallel multiplexed sSMLM-based spt-PALM of multiple subcellular organelles in live cells.

This project aims to develop a palette of bright switchable fluorescent probes for simultaneous multiplexed super-resolution imaging at the molecular level. We will increase the photon emission rate of the newly-designed switchable dyes and encode distinct spectral features of the switchable dyes in the two specific aims to collectively provide high resolution and multiplexity. This project will allow biomedical researchers, for the first time, to achieve simultaneous multi-color imaging of 6 molecular labels at 10-nm spatial resolution within 10 minutes. System performance will be investigated by imaging nanoscale engineered samples, and multiple molecular targets with distinct morphologies in cells.