Expansion Microscopy

膨胀显微镜

• 批准号: 10609512
• 负责人: Edward S. Boyden
• 金额: $ 60.53万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609512
• 关键词: Binding; Biological; Biological Preservation; Biology; Brain Mapping; Cells; Cellular Structures; Chemicals; Communities; Complex; Consumption; Cryoelectron Microscopy; Democracy; Development; Diamond; Disease; Educational workshop; Electron Microscopy; Equipment; Fluorescent Probes; Free Radicals; Goals; Grant; High temperature of physical object; Human; Hydrogels; Image; Internet; Label; Lipids; Maps; Medical; Methods; Microscope; Microscopy; Nucleic Acids; Paper; Paraffin Embedding; Persons; Polymers; Printing; Process; Proteins; Protocols documentation; RNA; Reagent; Research; Resolution; Resource Sharing; Sampling; Shapes; Specimen; Specimen Handling; Speed; Swelling; Technology; Time; Tissues; Training; Visualization; Water; Work; biological systems; cell type; cellular imaging; cold temperature; human tissue; imaging modality; insight; invention; nanoimaging; nanoscale; new therapeutic target; preservation; scale up; skills; superresolution microscopy; technology development; ultra high resolution

Biomolecules, such as nucleic acids, lipids, and proteins, are nanoscale in size, and often localized with nanoscale precision with respect to each other, and to cellular structures. Analyzing the nanoscale configurations of biomolecules in cells and tissues is critical for understanding how they work, as well as how they go wrong in disease states. Not surprisingly, much effort has been devoted to inventing methods (e.g., super-resolution microscopy, cryo-electron microscopy) for nanoimaging biological specimens, primarily in their preserved state. However, all of these technologies require expensive equipment, and specialized skillsets. Given that all biological systems involve nanoscale building blocks and their interactions, a major question is whether nanoimaging can be democratized, so that anyone could do it, without expensive equipment or extensive training. This grant is a first competitive renewal of our group’s primary grant that supports the development of a technology that we think could potentially meet this goal. We recently announced that in contrast to all previous methods for imaging preserved biological specimens, which magnify their images, specimens could themselves be physically magnified. This technology, which we call expansion microscopy (ExM), involves equipping key biomolecules or labels within a specimen with anchoring molecules, then densely and evenly permeating the biological specimen with a mesh of swellable polymer (that binds to the anchors, thus anchoring key biomolecules or labels to the polymer), softening the specimen to disrupt endogenous molecular interactions, and adding water to swell the polymer, which in turn pulls the biomolecules or labels apart from each other. The process is even down to the nanoscale, and thus enables nanoimaging of cells and tissues on ordinary microscopes. In addition, several recent papers point to an additional advantage of ExM – by pulling biomolecules apart from each other, you decrowd them for better labeling by fluorescent probes, sometimes turning invisible biomolecules into visible ones. ExM is already in use by many hundreds of research groups, with over 250 experimental preprints and papers appearing to date. Here we propose to make ExM simpler, more powerful, faster, more applicable to human samples, and more precise in resolution. Specifically, we will (Aim 1) create a unified, simple, high-speed ExM protocol; (Aim 2) create a unified, simple, high-speed, single-step 20x expansion protocol; (Aim 3) optimize the new unified, simple, and high-speed ExM protocols for human tissues. We propose a fast-paced, 4 year, technology development grant, with the goal of delivering, to the entire biology and medical community, a truly democratized toolbox that enables anyone to do nanoimaging. We will share all protocols as freely as possible both on the web and through protocol papers, as well as through hosting people at hands-on workshops.

生物分子，例如核酸，脂质和蛋白质，大小为纳米级，通常与 纳米级的精度相对于彼此和细胞结构。分析纳米级 细胞和组织中生物分子的构型对于了解它们的工作方式以及如何 他们在疾病状态下出错。毫不奇怪，已经大力致力于发明方法（例如， 超分辨率显微镜，冷冻电子显微镜），用于纳米影像生物标本，主要在其中 保存状态。但是，所有这些技术都需要昂贵的设备和专业的技能。 鉴于所有生物系统均涉及纳米级的构件及其相互作用，因此一个主要问题是 纳米影像是否可以民主化，以便任何人都可以做到，而无需昂贵的设备或 广泛的培训。这项赠款是我们集团主要赠款的首次竞争续约，该赠款支持 我们认为可能实现这一目标的技术的开发。我们最近宣布 与所有以前的成像保留的生物标本的方法形成对比，该方法放大了图像， 标本本身可能会被物理放大。我们称之为扩展显微镜的技术 （EXM），涉及将标本内的钥匙生物分子或标签与锚定分子装配，然后 密集，甚至用膨胀的聚合物网格渗透生物标本（与 锚点，从而将钥匙生物分子或标签锚定在聚合物上），使样品变软以破坏 内源分子相互作用，并加水以吞咽聚合物，进而拉动了聚合物 生物分子或彼此标签。该过程甚至取决于纳米级，因此可以实现 普通显微镜上细胞和组织的纳米影像学。此外，最近的几篇论文指出了 EXM的额外优势 - 通过将生物分子除外，您可以将它们删除以获得更好 通过荧光探针标记，有时将无形的生物分子变成可见的生物分子。 EXM已经在 迄今为止，数百个研究小组的使用，有250多个实验性预印本和论文。 在这里，我们建议使EXM更简单，更强大，更快，更适用于人类样本等等 精确的分辨率。特别是，我们将（AIM 1）创建一个统一，简单，高速EXM协议； （目标2） 创建一个统一，简单，高速，单步20倍扩展协议； （目标3）优化新的统一， 人体组织的简单和高速EXM方案。我们提出了一项快节奏的4年技术 开发赠款，目的是传递给整个生物学和医学界 民主化的工具箱，使任何人都可以进行纳米影像学。我们将尽可能免费共享所有协议 无论是在网络上还是通过协议论文，以及通过在动手讲习班上托管人员。