Collaborative Research: Spectral Discrimination of Single Molecules with Photoactivatable Fluorescence

在化学学部化学结构、动力学与机理- b （CSDM-B）和化学测量与成像（CMI）项目的支持下，迈阿密大学化学系的franisco M. Raymo和北卡罗莱纳州立大学纺织工程系的Yang Zhang正在开发一类具有光激活荧光的新型合成染料，用于生物成像应用。这项研究的目标是利用这些创新荧光染料的独特特性，以前所未有的分辨率来研究细胞过程，并最终贡献宝贵的化学工具来阐明正常细胞功能和疾病开始的基本因素。该项目位于有机化学和物理化学的界面，对生物学和医学具有长期影响。提议的活动是本科生、研究生和博士后助理的最佳训练场地，也包括通过两个机构现有的外展计划让高中生参与密集的实验室体验。该项目还将涉及两个机构中代表性不足的群体成员的参与，以及在当地社区大学的合作推广工作。本项目的目标是生成一个具有光激活荧光和可分辨单分子发射光谱的细胞渗透性和生物偶联性合成染料库。他们的设计基于光化学机制，以绿光激活硼二吡咯甲烷（BODIPY）荧光，这是该团队在之前的资助周期中开发的。它们的实现需要在两个平行的项目组件中对四个系列的光活化BODIPYs进行合成、结构表征、系综/单分子光谱分析和成像，这些BODIPYs的多环显色支架的环融合模式和/或取代基的性质不同。一个目的是解决光激活荧光的光谱位置和/或波段形状，以区分单分子发射光谱，以统计置信度进行结构区分。另一种方法是在发色平台上引入亲水基团和靶向配体，以选择性地标记和同时跟踪细胞内蛋白质。首要的科学目标是最终将结构、光化学和光物理特性的独特组合设计成创新的分子结构，从而实现对活细胞中蛋白质的前所未有的高通量多路单分子跟踪。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With support of the Chemical Structure, Dynamics & Mechanisms-B (CSDM-B) and Chemical Measurement and Imaging (CMI) Programs of the Chemistry Division, Françisco M. Raymo of the Department of Chemistry at the University of Miami and Yang Zhang of the Department of Textile Engineering at North Carolina State University are developing a new class of synthetic dyes with photoactivatable fluorescence for bioimaging applications. The goal of this research is to exploit the unique properties of these innovative fluorescent dyes to investigate cellular processes with unprecedented resolution and, ultimately, contribute invaluable chemical tools to elucidate the fundamental factors responsible for normal cellular functions and disease inception. The project lies at the interface of organic and physical chemistry with long-term implications in biology and medicine. The proposed activities are an optimal training ground for undergraduate, graduate students and postdoctoral associates as well as involve the participation of high-school students in intensive laboratory experiences through established outreach programs available at both institutions. The project will also involve participation of members of underrepresented groups at both institutions and collaborative outreach efforts at a local community college.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 as developed by the team in prior funding cycles. 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 overarching scientific goal is to ultimately engineer 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.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.