Expanding the HIDE nanoscopy toolbox: More organelles, colors, and modalities

项目摘要： 细胞生物学的一个主要目标是了解细胞的结构、相互作用、功能和调控 细胞器。今天，贝齐格、赫尔、莫尔纳、庄和现在的诺贝尔奖研究 许多其他的方法使科学家能够以 几十纳米。问题是图像只持续几秒钟，因此， 图片，无论多么美丽，隐藏着复杂的动力学和相互作用， 在健康和疾病中发挥作用。该提案扩展了双组分化学品的工具包， 一种名为“HIDE”（高密度环境敏感）的探测器， 可视化多个细胞器的动态，同时，在超分辨率， 前所未有的时代，和多种颜色。HIDE探针由两部分组成，一个细胞器- 具有反应性官能团（如反式环辛烯（TCO））特异性膜探针 和硅罗丹明染料（SiR，或在该提议中为HMSiR）与反应伴侣（例如， 四嗪）。这两个部分，当依次加入到活细胞中时， 连接反应，其将SiR染料以高密度定位在细胞器膜内。这里 HMSiR的光稳定性显著增强，并且可以产生单分子开关 (SMS)纳米图像比HMSiR标记的蛋白质长50倍。第一次隐藏 已经报道了探针（用于对高尔基体、内质网、线粒体和质膜成像 被我们在这里，我们将在这个强大的基础上进行构建，并扩展HIDE工具箱以包括探测器 四个额外的细胞器（早期，晚期和再循环内体和纤毛）（目的1），偶联 与正交SMS染料和化学品（目标2）所需的两个-并最终三个颜色 长期的SMS纳米显微镜。在最终目标（目标3）中，我们开发了HIDE-EM探头，以突出 离散细胞器的三维电子显微镜（EM）。每个目标都与可交付成果相关联 和时间相关的里程碑，并得到了重要的初步结果的支持。虽然我们 这里的重点是开发HIDE探针有用的SMS显微镜，只有微小的调整， 需要染料特性来产生用于STED的HIDE探针。技术 在此开发的将大大增强长时间推移活细胞的能力，范围和影响， 通过扩大可以单独评估的细胞器的数量和类型， 组合使用多种颜色，使用不同的成像模式，并在培养和 难以去除的原代细胞

Project Summary: A major goal of cell biology is to understand the structures, interactions, functions and regulation of cellular organelles. Today, Nobel-worthy research of Betzig, Hell, Moerner, Zhuang, and now many others allows scientists to visualize organelles in live cells at resolutions that number in the tens of nanometers. The problem is that the images last for only seconds, and thus the pictures, however beautiful, hide complex dynamics and interactions that underlie organelle function in health and disease. This proposal expands a toolkit of two-component chemical tools called ‘HIDE’ (High Density Environment-Sensitive) probes that will allow scientists to visualize the dynamics of multiple organelles, simultaneously, at super-resolution, for unprecedented times, and in multiple colors. HIDE probes consist of two parts, an organelle- specific membrane probe with a reactive functional group (such as trans-cyclooctene (TCO)) and a silicon rhodamine dye (SiR or, in this proposal, HMSiR) with a reaction partner (such as tetrazine). These two parts, when added sequentially to live cells, undergo an in situ tetrazine ligation reaction that localizes the SiR dye at high density within the organelle membrane. Here HMSiR photostability is significantly enhanced and can generate single molecule switching (SMS) nanoscopy images up to 50 times longer than HMSiR-tagged proteins. The first HIDE probes (used to image the Golgi, ER, mitochondria, and plasma membrane) have been reported by us. Here we build on this strong foundation and expand the HIDE toolbox to include probes for four additional organelles (early, late, and recycling endosomes, and cilia) (Aim 1), coupled with orthogonal SMS dyes and chemistries (Aim 2) required for two- and eventually three-color long-term SMS nanoscopy. In the final Aim (Aim 3), we develop HIDE-EM probes to highlight discrete organelles in 3D by electron microscopy (EM). Each Aim is associated with deliverables and time-dependent milestones and is supported by significant preliminary results. Although we focus here on developing HIDE probes useful for SMS microscopy, only minor adjustments in the dye identity would be needed to generate HIDE probes for STED. The technology developed herein will greatly enhance the power, scope, and impact of long time-lapse live cell nanoscopy by expanding the number and types of organelles that can be evaluated, alone, in combination using multiple colors, using different imaging modalities, and in both cultured and hard-to-transfect primary cells.