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

项目名称 用于高通量多路示踪活细胞内单分子的可光激活荧光团 项目摘要/摘要 我们项目的目标是开发同时具有光激活荧光的合成染料 在活细胞中追踪多种结构不同的细胞内成分。具体地说，拟议的研究 将导致实现可光激活的荧光团(PAF)的调色板，该调色板可以与 温和的绿色照明(&gt；500 nm)，在红色区域(&gt；600 nm)产生部分分辨的荧光 电磁波谱。它们的光激活条件将确保对活细胞的光损伤可以忽略不计， 相反，在操作现有PAF所需的严酷照射下，这是无法避免的。高潮 我们的PAF具有亮度、无限对比度和高耐光漂白性能，将使 单个光活化分子在纳米级(≤20 nm)的精确定位及其应用 基于单粒子跟踪的毫秒响应(≤10ms)数秒(≥1 S)跟踪 光活化定位显微镜(SPT-Palm)。它们的光谱分辨荧光将允许 通过在单分子水平上获取发射光谱来鉴定结构不同的探针， 依靠光谱单分子定位显微镜(SSMLM)。这样一个独特的组合 光化学和光物理特性是史无前例的，结合既定的战略 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和SPT-Palm的特征高通量对多个结构不同的目标进行监测 SSMLM的光谱分辨。我们的技术所能达到的空间分辨率是 传统的荧光成像协议及其高通量多路传输能力无法 在活细胞中应用到目前为止开发的许多合成染料和荧光蛋白。因此， 从拟议的研究中产生的创新的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 目前的荧光探头和成像方案无法访问。

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.