Photochemical Strategies to Activate Far-Red Fluorescence with Green Light

在该项目中，由化学部化学结构、动力学和机制B项目资助，迈阿密大学的Francisco Raymo教授和西北大学的Hao Zhang教授正在开发新的光化学和荧光方法来成像活细胞中的细胞内成分。这个具有挑战性的目标首先需要合成一系列结构相关的有机染料分子，这些分子被设计成可以进行由绿光引发的光反应。光反应使分子的一部分断裂，从而产生新的荧光团，或发光分子，发出红光。仔细的结构设计也允许对细胞内成分进行位点特异性标记。由此产生的荧光团通过使用高分辨率荧光显微镜，具有纳米级的空间分辨率，可以对活细胞中的许多亚细胞成分进行成像。这些基础研究的目标之一是确定最佳的结构设计，将非荧光反应物在温和的绿光照射下转化为荧光产物，而绿光的波长对活细胞无害。该项目预计将对光化学和荧光成像产生直接影响。创造创新的化学工具，以前所未有的分辨率来研究活细胞结构，在生物学和医学方面具有更广泛的意义。这些研究活动为参与的博士后、研究生和本科生提供了良好的培训机会。他们在化学合成、光谱分析和荧光成像方面发展实验室专业知识，并学习如何培养和操作活细胞。在这个过程中，他们可以提高自己的沟通和表达能力，同时也有机会指导高中生和本科生进行密集的实验室体验。该项目加强了与迈阿密戴德学院现有的学生交换项目，该项目为来自科学领域未被充分代表的群体的学生提供研究经验。该项目的研究活动旨在开发一种通用的敏化机制，以诱导绿光的光化学反应，从而为生物应用提供具有最佳性能的各种光解保护基团。新的光化学是基于对BODIPY（硼-二吡咯甲烷）-恶嗪染料的修饰，它的恶嗪成分经过光裂解。染料结构包括光谱调整的设计元素，增强水溶解度和位点特异性附着于细胞。绿光（500 - 530nm）是一种对生物样品无害的波长，通过产生新的荧光团，使明亮的远红光荧光成像成为可能。这些基础研究提供了光激活荧光的结构设计，在与活细胞兼容的照射条件下，使用单分子定位显微镜在单分子水平上以纳米级精度同时定位多个光谱不同的荧光团。该化合物具有独特的结构、光化学和光物理性质的组合，可以转化为具有优越性能的探针，超过迄今为止开发的用于活细胞亚衍射成像的少数光活化合成染料。因此，这个研究项目，主要针对光切换荧光结构的化学合成和光谱分析，预计将对光化学和荧光成像的变革意义产生直接影响。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。

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.