Photoactivatable Fluorophores for High-Throughput Multiplexed Tracking of Single-Molecules in Live Cells

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PROJECT TITLE Photoactivatable Fluorophores for High-Throughput Multiplexed Tracking of Single-Molecules in Live Cells PROJECT ABSTRACT/SUMMARY The goal of our project is to develop synthetic dyes with photoactivatable fluorescence for the simultaneous tracking of multiple structurally-distinct intracellular components in live cells. Specifically, the proposed studies will lead to the realization of a palette of photoactivatable fluorophores (PAFs) that can be photoactivated with mild green illumination (>500 nm) to produce partially-resolved fluorescence across the red region (>600 nm) of the electromagnetic spectrum. Their photoactivation conditions will ensure negligible photodamage to live cells, which instead cannot be avoided under the harsh irradiation required to operate existing PAFs. The high brightness, infinite contrast and high photobleaching resistance engineered into our PAFs will enable the localization of individual photoactivated molecules with precision at the nanometer level (≤20 nm) and their tracking with millisecond response (≤10 ms) for several seconds (≥1 s) on the basis of single-particle tracking photoactivated localization microscopy (spt-PALM). Their spectrally-resolved fluorescence will permit the identification of structurally-distinct probes with the acquisition of emission spectra at the single-molecule level, relying on spectroscopic single-molecule localization microscopy (sSMLM). Such a unique combination of photochemical and photophysical properties is unprecedented and, in conjunction with established strategies to label selectively different intracellular components of live cells with synthetic dyes, will allow the simultaneous monitoring of multiple structurally-distinct targets with the characteristic high-throughput of spt-PALM and spectral discrimination of sSMLM. The spatial resolution possible with our technology cannot be achieved with conventional fluorescence imaging protocols and its high-throughput multiplexing capabilities cannot be implemented in live cells with the many synthetic dyes and fluorescent proteins developed so far. Thus, the innovative synthetic constructs that will emerge from the proposed studies can contribute to the investigation of the fundamental factors governing cellular processes with multiplexing and super-resolution capabilities that are not accessible with current fluorescent probes and imaging schemes.