Advanced Probes and Targeting for Multiscale Microscopy

描述（由申请人提供）：本课题旨在改进和利用荧光蛋白（FPs）；量子点（QDs），将量子点等纳米颗粒递送到细胞质目标的方法，以及相关的光学/电子显微镜（EM）成像技术。通过创建非常大的、多样化的遗传变异文库，并在模拟单分子成像的条件下在固定化活细胞中筛选它们的光物理稳定性，将改善FPs的光稳定性。特别强调的是在细胞自身荧光最小的长波长的FPs发射，以及共享激发最大但同时发射不同波长的多色超分辨率定位的FPs。优化出尺寸小但发射波长长的量子点、激发和发射最大值之间间隙大的量子点以及可光开关的量子点。为了帮助将量子点传递到细胞质目标，将通过噬菌体展示进化出从内体释放纳米颗粒货物的肽，然后将其应用于量子点。一旦量子点进入细胞质，它们将通过双砷-四胱氨酸配对或半胱甘肽-单链抗体复合物与感兴趣的标记蛋白结合。相关光学/EM成像对于结合活细胞动力学和更高的空间分辨率，包括细胞骨架和细胞器在内的细胞背景，是非常有价值的。这种相关成像将通过开发产生单线态氧的FPs来触发em可见纳米沉淀物的形成，通过改进阴极发光，即通过电子束激发荧光检测FPs和量子点，以及通过优化易于区分大小和形状的量子点来推进。为了验证新的探针和技术，关键亚细胞过程的功能动力学将使用Co-PI实验室目前正在研究的模型系统进行研究。其中包括“中介复合体”——一种保守的多亚基复合体，它调节酵母、小鼠和人类的转录机制，以及有丝分裂过程中高尔基体和中心粒等细胞器的分子标记动力学。公共卫生相关性：显微成像是将遗传学、生物化学、生理学和显微解剖学整合成细胞功能连贯图像的最佳方法之一，特别是当感兴趣的关键特定蛋白质可以被标记以使其独特可见时。本提案的总体目标是提高通用方法的通用性、检测灵敏度和空间分辨率，以成像细胞内几乎任何所需蛋白质的动态位置和功能，无论是在正常条件下还是在疾病过程中。

DESCRIPTION (provided by applicant): This proposal aims to improve and exploit fluorescent proteins (FPs); quantum dots (QDs), methods for delivery of nanoparticles such as QDs to cytoplasmic targets, and techniques for correlative optical/electron microscopic (EM) imaging. Photostability of FPs will be improved by creating very large, diverse libraries of genetic variants and screening them in immobilized live cells for photophysical stability under conditions mimicking single molecule imaging. Particular emphasis will be placed on FPs emitting at long wavelengths where cellular autofluorescence is minimal, and FPs that share excitation maxima but emit at different wavelengths for simultaneous multicolor hyper-resolution localization. QDs with small size but long emission wavelengths, QDs with large gaps between excitation and emission maxima, and photoswitchable QDs will be optimized. To help deliver QDs to cytoplasmic targets, peptides that release nanoparticle cargoes from endosomes will be evolved by phage display then applied to QDs. Once QDs have entered the cytoplasm, they will bind to tagged proteins of interest either via biarsenical-tetracysteine pairing or hapten-single-chain antibody complexes. Correlative optical/EM imaging is extremely valuable for combining live cell dynamics with yet higher spatial resolution, including cellular context including cytoskeleton and organelles. Such correlative imaging will be advanced by developing FPs that generate singlet oxygen to trigger formation of EM-visible nanoprecipitates, by improving cathodoluminescence, i.e. detection of FPs and QDs by electron-beam-excited fluorescence, and by optimizing QDs of readily distinguishable sizes and shapes. To validate the new probes and technologies, functional dynamics of key subcellular processes will be studied using model systems currently under study in the labs of the Co-PI's. These include the "Mediator complex" - a conserved multi- subunit complex which regulates the transcriptional machinery in yeast, mouse, and humans, and dynamics of molecular markers of organelles like the Golgi apparatus and centriole during mitosis. Public Health Relevance: Microscopic imaging is one of the best ways to integrate genetics, biochemistry, physiology, and microanatomy into a coherent picture of cell function, especially when key specific proteins of interest can be tagged to make them uniquely visible. The overall goal of this proposal is to improve the versatility, detection sensitivity, and spatial resolution of general methods to image the dynamic location and function of nearly any desired protein(s) inside cells, both under normal conditions and during disease processes.