Photoactivatable Fluorophores for High-Throughput Multiplexed Tracking of Single-Molecules in Live Cells

用于活细胞中单分子高通量多重追踪的光活化荧光团

• 批准号: 10794007
• 负责人: Francisco M Raymo
• 金额: $ 9.05万
• 依托单位: UNIVERSITY OF MIAMI CORAL GABLES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10794007
• 关键词: Biomedical Research; Cell physiology; Cells; Characteristics; Development; Discrimination; Dyes; Electromagnetics; Engineering; Ensure; Fluorescence; Fluorescent Probes; Goals; Image; Individual; Investigation; Label; Lighting; Microscopy; Molecular; Monitor; Photobleaching; Property; Proteins; Protocols documentation; Resistance; Resolution; Scheme; Technology; Visualization; design; fluorescence imaging; fluorophore; innovation; irradiation; millisecond; nanometer; optical spectra; particle; photoactivation; response; single molecule; spatiotemporal; synthetic construct; ultra high resolution

PROJECT TITLE Photoactivatable Fluorophores for High-Throughput Multiplexed Tracking of Single-Molecules in Live Cells PROJECT ABSTRACT/SUMMARY The goal of our project is to develop synthetic dyes with photoactivatable fluorescence for the simultaneous tracking of multiple structurally-distinct intracellular components in live cells. Specifically, the proposed studies will lead to the realization of a palette of photoactivatable fluorophores (PAFs) that can be photoactivated with mild green illumination (>500 nm) to produce partially-resolved fluorescence across the red region (>600 nm) of the electromagnetic spectrum. Their photoactivation conditions will ensure negligible photodamage to live cells, which instead cannot be avoided under the harsh irradiation required to operate existing PAFs. The high brightness, infinite contrast and high photobleaching resistance engineered into our PAFs will enable the localization of individual photoactivated molecules with precision at the nanometer level (≤20 nm) and their tracking with millisecond response (≤10 ms) for several seconds (≥1 s) on the basis of single-particle tracking photoactivated localization microscopy (spt-PALM). Their spectrally-resolved fluorescence will permit the identification of structurally-distinct probes with the acquisition of emission spectra at the single-molecule level, relying on spectroscopic single-molecule localization microscopy (sSMLM). Such a unique combination of photochemical and photophysical properties is unprecedented and, in conjunction with established strategies to label selectively different intracellular components of live cells with synthetic dyes, will allow the simultaneous monitoring of multiple structurally-distinct targets with the characteristic high-throughput of spt-PALM and spectral discrimination of sSMLM. The spatial resolution possible with our technology cannot be achieved with conventional fluorescence imaging protocols and its high-throughput multiplexing capabilities cannot be implemented in live cells with the many synthetic dyes and fluorescent proteins developed so far. Thus, the innovative synthetic constructs that will emerge from the proposed studies can contribute to the investigation of the fundamental factors governing cellular processes with multiplexing and super-resolution capabilities that are not accessible with current fluorescent probes and imaging schemes.

项目标题 可光活化的荧光团，用于活细胞中单分子的高通量多路复用跟踪 项目摘要/摘要 我们项目的目的是与同时使用光活化荧光开发合成染料 跟踪活细胞中多个结构上赋予的细胞内组件。具体而言，拟议的研究 将导致可以通过光活化的光活化荧光团（PAF）的调色板实现 温和的绿色照明（> 500 nm），可在红色区域（> 600 nm）中部分分辨荧光 他们的光活化条件将确保对活细胞的光损伤可忽略 相反，在操作现有PAF所需的HARMSH辐射下无法避免这种情况。高 亮度，无限的对比和高光漂白的阻力，将其设计为我们的PAF 在纳米水平（≤20nm）及其其精度的单个光活化分子及其定位 根据单粒子跟踪，用毫秒响应（≤10ms）进行几秒钟（≥1s）的跟踪 光活化的定位显微镜（SPT-PALM）。它们的光谱分辨荧光将允许 鉴定在单分子水平上获取发射光谱的结构存在问题， 依靠光谱单分子定位显微镜（SSMLM）。如此独特的组合 光化学和光物理特性是前所未有的，并结合了既定的策略 选择性地标记具有合成染料的活细胞的不同细胞内组件，将允许简单 用SPT-PALM和 SSMLM的频谱区分。通过我们的技术无法实现的空间分辨率 常规的荧光成像协议及其高通量多路复用功能不能为 到目前为止，在活细胞中实施了许多合成染料和荧光蛋白。那， 拟议研究将出现的创新合成结构可以有助于研究 具有多重和超分辨率功能的细胞过程的基本因素是 目前的荧光问题和成像方案无法访问。