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

项目名称 光激活荧光团对活细胞中单分子的高通量多重追踪 项目摘要/总结 我们的目标是开发具有光激活荧光的合成染料，用于同时 跟踪活细胞中多种结构不同的细胞内成分。具体而言，拟议的研究 将导致实现可光活化的荧光团（PAF）的调色板，其可以被光活化， 温和的绿色照明（>500 nm），以产生跨越红色区域（>600 nm）的部分分辨荧光， 电磁波谱它们的光活化条件将确保对活细胞的光损伤可忽略不计， 而这在操作现有PAF所需的苛刻辐射下是无法避免的。高 亮度、无限对比度和高抗光漂白性将使我们的PAF 在纳米级（≤20 nm）精确定位单个光活化分子， 在单粒子跟踪的基础上，以毫秒级响应（≤10 ms）跟踪数秒（≥1 s） 光活化定位显微术（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）。如此独特的组合， 光化学和生物物理特性是前所未有的，并结合既定的战略， 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的特征性高通量监测多个结构上不同的靶标， sSMLM的光谱鉴别。我们的技术所能达到的空间分辨率， 传统的荧光成像协议及其高通量多路复用能力不能 在活细胞中使用迄今为止开发的许多合成染料和荧光蛋白实现。因此 拟议研究中出现的创新合成构建体可以有助于研究 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， 不能用当前的荧光探针和成像方案获得。

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.