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.

项目名称 用于活细胞中单分子高通量多重追踪的光活化荧光团 项目摘要/总结 我们项目的目标是开发具有光活化荧光的合成染料，用于同时 具体来说，所提出的研究跟踪活细胞中多种结构不同的细胞内成分。 将导致实现一系列可光活化荧光团（PAF），可以用以下物质进行光活化 温和的绿色照明（> 500 nm）可在红色区域（> 600 nm）产生部分分辨的荧光 它们的光活化条件将确保对活细胞的光损伤可以忽略不计， 相反，在运行现有 PAF 所需的严酷辐射下，这是不可避免的。 我们的 PAF 具有亮度、无限对比度和高抗光漂白性，将使 单个光激活分子的精确定位在纳米级（≤20 nm）及其 在单粒子跟踪的基础上实现毫秒级响应（≤10 ms）跟踪数秒（≥1 s） 光激活定位显微镜 (spt-PALM) 的光谱分辨荧光将允许 通过获取单分子水平的发射光谱来识别结构不同的探针， 依靠光谱单分子定位显微镜（sSMLM）这种独特的组合。 光化学和光物理特性是前所未有的，并且结合既定策略 用合成染料选择性地标记活细胞的不同细胞内成分，将允许同时 利用 spt-PALM 的高通量特性监测多个结构不同的目标 sSMLM 的光谱辨别能力无法通过我们的技术实现。 传统的荧光成像协议及其高通量多重功能无法 迄今为止，已开发出多种合成染料和荧光蛋白，可在活细胞中实现。 拟议研究中出现的创新合成结构可以有助于研究 控制具有多路复用和超分辨率能力的细胞过程的基本因素是 当前的荧光探针和成像方案无法实现。