Expansion Microscopy

膨胀显微镜

• 批准号: 9301863
• 负责人: Edward S. Boyden
• 金额: $ 57.4万
• 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 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9301863
• 关键词: 3-Dimensional; Antibodies; Axon; Binding; Biological; Biology; Biotechnology; Brain; Caenorhabditis elegans; Cells; Chemicals; Chemistry; Clinical; DNA; Dimensions; Disease; Educational workshop; Engineering; Equipment; Fluorescent in Situ Hybridization; Gel; Generations; Grant; Image; Imagery; Individual; Investigation; Isotropy; Label; Learning; Lipids; Manuscripts; Mechanics; Methods; Microscope; Microscopy; Molecular; Nature; Nervous system structure; Neurosciences; Nucleic Acids; Optics; Organ; Organism; Pancreas; Pattern; Polymers; Proteins; Protocols documentation; Publishing; RNA; Research; Resolution; Running; Sampling; Science; Slice; Specimen; Structure; Synapses; Technology; Testing; Therapeutic; Thick; Time; Tissue imaging; Tissues; Validation; Virus; Water; Zebrafish; cellular imaging; complex biological systems; design; fluorophore; insight; interest; lens; light microscopy; mechanical properties; molecular scale; nanoscale; novel; novel strategies; technology development; tool

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.

生物学和神经科学中的许多问题将大大受益于一种使分子生物学成为可能的技术。 信息（例如，特定核酸和蛋白质的身份）在整个保存的3-D成像 样本（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 2015年发表在《科学》杂志上的一项研究：与早期的光学显微镜放大方法相比， 它依靠透镜来光学放大细胞和组织的图像， 标本通过直接在样品内机械地合成可溶胀的凝胶， 将标本浸泡，然后在水中透析，我们可以将组织在线性尺寸上扩大约4.5倍。 这种方法可以将位于衍射极限体积内的分子分离到足够大的距离， 可以用传统的显微镜进行分辨，有效分辨率约为70 nm。我们称之为小说 方法扩展显微镜（ExM）。从那时起，我们使这项技术更容易使用，创造了一个 ExM的一个版本，我们称之为proExM（蛋白质保留ExM），它使用市售化学品， 将遗传编码的荧光团或抗体携带的荧光团直接锚到可溶胀凝胶上，和 验证其在各种组织中保留纳米级特征的能力（在Nature Biotechnology接受） 并将ExM扩展到RNA分子的锚定和扩张， RNA可视化，我们称之为ExFISH（在Nature Methods接受）。有大量被压抑的需求， 一种用于扩展三维标本的纳米级成像方法，尤其是不需要专门的 设备;我们每周在麻省理工学院的团队中接待来访者来学习和练习ExM， Janelia研究校园，我们将在2016年8月举办一个研讨会，教授ExM实践。考虑到潜在的 对于ExM解决神经科学中的许多问题，我们现在建议增加其功能和多功能性。 具体来说，我们将（目标1）开发优化形式的ExM困难的标本（如C。elegans），as 以及单样本验证策略（通过在样本中创建“物理比例尺”），（目标2）发明 新的化学成分，可将样品的线性尺寸扩大20倍或80倍， 有效的分辨率分别，和（目的3）扩展ExM锚定化学的可视化脂质 和DNA，以及生物分子的组合（例如，同时看到蛋白质、DNA和RNA）。我们 该项目将产生在神经科学以及整个生物学中非常适用的工具。我们提出了一个 快节奏，4年的技术开发赠款，将导致工具，将使大量的 需要分析的科学问题。我们将尽可能免费地分发所有工具，并教授其使用方法。