Expansion Microscopy

生物学和神经科学中的许多问题将极大地受益于使分子 信息(例如，特定核酸和蛋白质的身份)将在整个保存的3-D中成像 样品(例如，大脑电路)，具有纳米级的精度。因此，我们开发了一种全新的 方法，发表在2015年的《科学》杂志上：与早期的光学显微镜放大方法不同， 它依靠透镜光学放大细胞和组织的图像，我们物理上放大保存下来的 标本。通过直接在样品中机械地合成可膨胀聚电解质凝胶 将标本均质，然后在水中透析，可以使组织在线性尺寸上膨胀~4.5倍。 这种方法可以将位于衍射限制体积内的分子分离到足够大的距离 用常规显微镜可以分辨，有效分辨率为~70 nm。我们称这部小说为 方法扩张显微镜(EXM)。从那时起，我们使这项技术更易于使用，创造了一种 Exm的版本，我们称之为proExM(蛋白质保留exm)，它使用商业上可获得的化学物质来 将基因编码的荧光团或抗体携带的荧光团直接锚定到可膨胀凝胶上；以及 验证其在各种组织中保留纳米级特征的能力(自然生物技术接受) 并将exm扩展到纳米级的rna分子之间的锚定和膨胀。 RNA可视化，我们称之为ExFISH(自然方法接受)。有巨大的被压抑的需求，对 用于扩展的3-D样品的纳米尺度成像方法，尤其是不需要专门的 设备；我们每周在麻省理工学院的小组中接待来访者，他们来学习和练习exm，并与 珍妮莉亚研究校园我们将在2016年8月举办一个工作坊，教授exm实践。考虑到潜在的 为了解决神经科学中的许多问题，我们现在建议增加它的功能和通用性。 具体地说，我们将(目标1)为困难的标本(如线虫)开发优化的exm形式，如 以及单一样本验证的策略(通过在样本中创建“物理标尺”)，(目标2)发明 新的化学成分使样品在线性尺寸上扩展20倍或80倍，实现~15 nm和~3 nm 有效分辨率和(目标3)扩展EXM锚定化学以显示脂类 和DNA，以及生物分子组合(例如，同时看到蛋白质、DNA和RNA)。我们的 该项目将产生在神经科学和整个生物学中都有很大适用性的工具。我们提出了一个 快节奏、为期4年的技术开发拨款，将导致工具支持大量 需要分析的科学问题。我们将尽可能自由地分发所有工具，并教授其使用方法。

Many questions in biology and neuroscience would benefit greatly from a technology that enabled molecular information (e.g., the identities of specific nucleic acids and proteins) to be imaged throughout preserved 3-D specimens (e.g., brain circuits), with nanoscale precision. Accordingly, we developed a fundamentally new approach, published in Science in 2015: in contrast to earlier methods of magnification in light microscopy, which rely on lenses to optically magnify images of cells and tissues, we physically magnify preserved specimens. By synthesizing a swellable polyelectrolyte gel directly within a specimen, mechanically homogenizing the specimen, then dialyzing in water, we could expand tissues by ~4.5x in linear dimension. This method could separate molecules located within a diffraction-limited volume to distances great enough to be resolved with conventional microscopes, resulting in an effective resolution of ~70 nm. We call this novel method expansion microscopy (ExM). Since then, we have made the technology easier to use, creating a version of ExM which we call proExM (protein retention ExM) that uses commercially available chemicals to directly anchor genetically encoded fluorophores or antibody-borne fluorophores to the swellable gel, and validating its ability to preserve nanoscale features in a variety of tissues (accepted at Nature Biotechnology) and extended ExM to the anchoring and expansion of RNA molecules away from one another for nanoscale RNA visualization, which we call ExFISH (accepted at Nature Methods). There is great pent-up demand for a method of nanoscale imaging for extended 3-D specimens, especially one that requires no specialized equipment; we host visitors weekly in our group at MIT to come and learn and practice ExM, and with the Janelia Research Campus we will run a workshop to teach ExM hands-on in August 2016. Given the potential for ExM to solve many problems in neuroscience, we now propose to increase its power and versatility. Specifically, we will (Aim 1) develop optimized forms of ExM for difficult specimens (such as C. elegans), as well as strategies for single-sample validation (by creating “physical scalebars” within samples), (Aim 2) invent new chemistries for expanding specimens by 20x or 80x in linear dimension, enabling ~15 nm and ~3 nm effective resolutions respectively, and (Aim 3) extend ExM anchoring chemistries for the visualization of lipids and DNA, as well as combinations of biomolecules (e.g., seeing proteins, DNA, and RNA all at once). Our project will result in tools of great applicability in neuroscience, as well as throughout biology. We propose a fast-paced, 4 year technology development grant that will result in tools that will enable a large number of scientific problems to be analyzed. We will distribute all tools as freely as possible, and teach usage thereof.