Collaborative Research: Spectral Discrimination of Single Molecules with Photoactivatable Fluorescence

在化学系化学结构、动力学和机理B(CSDM-B)和化学测量和成像(CMI)计划的支持下，迈阿密大学化学系的FrançIsco M.Raymo和北卡罗来纳州立大学纺织工程系的Yang Zhang正在开发一种新型的具有光激活荧光的合成染料，用于生物成像应用。这项研究的目标是利用这些创新的荧光染料的独特性质，以前所未有的分辨率研究细胞过程，并最终贡献宝贵的化学工具来阐明导致正常细胞功能和疾病发生的基本因素。该项目位于有机化学和物理化学的交界处，具有生物学和医学的长期影响。拟议的活动是本科生、研究生和博士后助理的最佳培训场所，并通过这两个机构提供的现有外联计划，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群体的成员参与，以及在当地社区大学的合作外联工作。该项目的目标是建立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可结合合成染料资料库。他们的设计是基于该团队在之前的资金周期中开发的用绿光激活BODIPY(BODIPY)荧光的光化学机制。它们的实现需要在两个平行的项目组件中合成、结构表征、系综/单分子光谱分析和成像四个系列的可光激活的BODIPY，它们的多环发色支架的环-融合模式和/或其取代基的性质不同。一种是解决光激活荧光的光谱位置和/或谱带形状，以充分区分单分子发射光谱，从而以统计置信度进行结构区分。另一种是在发色平台上引入亲水基团和靶向配基，选择性地标记和跟踪细胞内蛋白质。总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构造，以实现对活细胞中蛋白质的前所未有的高通量多路复用单分子跟踪。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.