Photochemical Strategies to Activate Far-Red Fluorescence with Green Light

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In this project, funded by the Chemical Structure, Dynamics & Mechanisms B Program of the Chemistry Division, Professor Francisco Raymo at the University of Miami and Professor Hao Zhang at Northwestern University are developing new photochemistry and fluorescence methods to image intracellular components in live cells. This challenging objective first requires the synthesis of a series of structurally related organic dye molecules, which are designed to undergo photoreactions initiated by green light. Photoreaction breaks off a portion of the molecule, thereby generating a new fluorophore, or light emitting molecule, which emits red light. Careful structure design also permits site-specific labeling of intracellular components. The resulting fluorophores enable the imaging of many sub-cellular components in live cells by using high resolution fluorescence microscopy, with spatial resolution at the nanometer level. One goal of these fundamental studies is the identification of optimal structural designs to convert a non-fluorescent reactant into a fluorescent product under mild illumination with green light, a wavelength that is innocuous to live cells. This project is expected to have an immediate impact on photochemistry and fluorescence imaging. Creation of innovative chemical tools to investigate live-cell structures with unprecedented resolution has broader implications in biology and medicine. These research activities provide an outstanding training opportunity for the participating postdoctoral associate, graduate students and undergraduate students. They develop laboratory expertise in chemical synthesis, spectroscopic analysis and fluorescence imaging as well as learn how to culture and manipulate live cells. In the process, they can refine their communication and presentation skills, while also having the opportunity to supervise high-school and undergraduate students in intensive laboratory experiences. This project reinforces an existing student-exchange program with Miami-Dade College, which provides research experiences for students from groups underrepresented in science.Research activities in this project are aimed at the development of a general sensitization mechanism to induce photochemical reactions with green light, thereby providing access to a diverse collection of photocleavable protecting groups with optimal properties for biological applications. New photochemistry is based on modifications to BODIPY (boron-dipyrromethene)-oxazine dyes, which undergo photocleavage of the oxazine component. Dye structures include design elements for spectral tuning, enhancement of aqueous solubility and site-specific attachment to cells. Photoreactions with green light (500 – 530 nm), a wavelength harmless to biological samples, enable bright far-red light fluorescence imaging by generating a new fluorophore. These fundamental studies contribute structural designs to photoactivate fluorescence, under irradiation conditions compatible with live cells, and simultaneously localize multiple spectrally-distinct fluorophores at the single-molecule level with nanoscale precision using single molecule localization microscopy. The unique combination of structural, photochemical and photophysical properties engineered into the proposed compounds can translate into probes with superior performance over the few photoactivatable synthetic dyes developed so far for sub-diffraction imaging of live cells. Thus, this research project, predominantly aimed at the chemical synthesis and spectroscopic analysis of photoswitchable fluorescent constructs, is expected to have an immediate impact on photochemistry and transformative implications in fluorescence imaging.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.