Advanced Probes and Targeting for Multiscale Microscopy

描述（由申请人提供）：本提案旨在改进和利用荧光蛋白（FP）;量子点（QD），将纳米颗粒（如QD）递送至细胞质靶点的方法，以及相关光学/电子显微镜（EM）成像技术。FP的光稳定性将通过创建非常大的、多样化的遗传变体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生物物理稳定性来改善。特别强调的是将放在FP发射在长波长的细胞自发荧光是最小的，和FP共享激发最大值，但发射在不同的波长，同时超分辨定位。具有小尺寸但长发射波长的QD、在激发和发射最大值之间具有大间隙的QD以及光可切换QD将被优化。为了帮助将QD递送至细胞质靶标，从内体释放纳米颗粒货物的肽将通过噬菌体展示进化，然后应用于QD。一旦量子点进入细胞质，它们将通过双砷-四半胱氨酸配对或半抗原-单链抗体复合物与标记的目标蛋白质结合。相关光学/EM成像对于将活细胞动力学与更高的空间分辨率相结合是非常有价值的，包括细胞背景，包括细胞骨架和细胞器。通过开发产生单线态氧以触发EM可见纳米沉淀物的形成的FP，通过改善阴极发光，即通过电子束激发的荧光检测FP和QD，以及通过优化易于区分的尺寸和形状的QD，将推进这种相关成像。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。这些包括“介体复合物”-一种保守的多亚基复合物，其调节酵母、小鼠和人类中的转录机制，以及有丝分裂期间细胞器如高尔基体和中心粒的分子标记物的动力学。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到细胞功能的连贯图像中的最佳方法之一，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。该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，以在正常条件下和疾病过程中对细胞内几乎任何所需蛋白质的动态位置和功能进行成像。

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.