Photochemical Strategies to Activate Far-Red Fluorescence with Green Light

用绿光激活远红荧光的光化学策略

• 批准号: 1954430
• 负责人: Francisco Raymo
• 金额: $ 58万
• 依托单位: University of Miami
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1954430&HistoricalAwards=false
• 关键词: Photochemical; Strategies; Activate; Far; Red

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.

在该项目中，由化学系化学结构，动力学机制B计划资助，迈阿密大学的弗朗西斯科教授和西北大学的张浩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组分。这一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，这些分子被设计为经历由绿色光引发的光反应。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，其发出红光。仔细的结构设计还允许细胞内组分的位点特异性标记。 由此产生的荧光团能够通过使用高分辨率荧光显微镜对活细胞中的许多亚细胞组分进行成像，其空间分辨率为纳米级。这些基础研究的一个目标是确定最佳结构设计，以在绿色光（对活细胞无害的波长）的温和照射下将非荧光反应物转化为荧光产物。 预计该项目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助理，研究生和本科生提供了一个出色的培训机会。 他们发展化学合成，光谱分析和荧光成像方面的实验室专业知识，并学习如何培养和操纵活细胞。 在这个过程中，他们可以完善自己的沟通和演示技巧，同时也有机会监督高中和本科生在密集的实验室经验。 该项目加强了与迈阿密-戴德学院现有的学生交流项目，该项目为来自科学领域代表性不足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，以诱导与绿色光的光化学反应，从而提供获得具有生物应用最佳特性的多种光可裂解保护基团的机会。新的光化学是基于对BODIPY（硼-二吡咯亚甲基）-恶嗪染料的修饰，其经历恶嗪组分的光裂解。 染料结构包括用于光谱调谐、水溶性增强和与细胞的位点特异性附着的设计元素。 与绿色光（500 - 530 nm）（对生物样品无害的波长）的光反应通过产生新的荧光团而实现明亮的远红光荧光成像。 这些基础研究有助于光激活荧光的结构设计，在与活细胞相容的照射条件下，并同时使用单分子定位显微镜以纳米级精度在单分子水平上定位多个光谱不同的荧光团。 设计到所提出的化合物中的结构、光化学和光物理性质的独特组合可以转化为具有比迄今为止开发的用于活细胞的亚衍射成像的少数可光活化的合成染料更优越的上级性能的探针。 因此，该研究项目，主要针对光开关荧光结构的化学合成和光谱分析，预计将对光化学产生直接影响，并在荧光成像中产生变革性影响。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

项目成果

Photoactivatable Fluorophores for Bioimaging Applications

用于生物成像应用的光活化荧光团

• DOI: 10.1021/acsaom.3c00025
• 发表时间: 2023
• 期刊: ACS Applied Optical Materials
• 作者: Zhang, Yang;Zheng, Yeting;Tomassini, Andrea;Singh, Ambarish Kumar;Raymo, Françisco M.
• 通讯作者: Raymo, Françisco M.