Expansion Microscopy

生物分子，如核酸、脂质和蛋白质，在尺寸上是纳米级的，并且通常被局部化， 纳米级的精确度，以及细胞结构。分析纳米尺度 细胞和组织中生物分子的构型对于理解它们如何工作以及它们如何 它们在疾病状态下会出错。毫不奇怪，已经投入了很多努力来发明方法（例如， 超分辨率显微镜、低温电子显微镜），用于对生物样本进行纳米成像，主要是在其 保存状态然而，所有这些技术都需要昂贵的设备和专业技能。 鉴于所有生物系统都涉及纳米级的构建模块及其相互作用，一个主要问题是 纳米成像是否可以普及，让任何人都可以做，而不需要昂贵的设备， 广泛的训练。这项赠款是我们集团的主要赠款的第一次竞争性更新， 我们认为有可能实现这一目标的技术。我们最近宣布，在 与之前所有用于对保存的生物标本进行成像的方法（放大其图像）相比， 标本本身可以被物理放大。这项技术，我们称之为扩张显微镜 (ExM)，涉及在样本内配备锚定分子的关键生物分子或标签，然后 用可溶胀聚合物网（其结合到生物样品上）致密且均匀地渗透生物样品， 锚，从而将关键的生物分子或标记锚定到聚合物上），软化样本以破坏 内源性分子相互作用，并加入水以溶胀聚合物，这反过来又拉动聚合物。 生物分子或标记彼此分开。该工艺甚至可以达到纳米级，因此能够 在普通显微镜上对细胞和组织进行纳米成像。此外，最近的几篇论文指出， ExM的另一个优点-通过将生物分子彼此分开， 用荧光探针标记，有时把看不见的生物分子变成可见的。ExM已经在 被数百个研究小组使用，迄今已有250多篇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，以及更多 分辨率精确。具体来说，我们将（目标1）创建一个统一的，简单的，高速的ExM协议;（目标2） 创建统一、简单、高速、单步20倍扩展协议;（目标3）优化新的统一， 用于人体组织的简单高速ExM协议。我们提出了一个快节奏，4年，技术 发展赠款，目标是向整个生物学和医学界提供一个真正的 使任何人都能进行纳米成像的民主化工具箱。我们将尽可能自由地分享所有协议 在网上和通过议定书文件以及通过在实践研讨会上接待人们。

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.