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.

生物学和神经科学方面的许多问题将从启用分子的技术中受益匪浅 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 标本（例如，脑电路），具有纳米级精度。根据，我们开发了一种从根本上开发的 方法于2015年发表在科学上：与光学显微镜中的早期放大方法相反， 依靠镜片光学地放大细胞和组织的图像，我们物理放大了。 标本。通过直接在样品中合成可膨胀的聚电解质凝胶，机械地 将样品匀浆，然后在水中进行透析，我们可以在线性尺寸上扩展组织约4.5倍。 该方法可以将位于衍射限量的体积内的分子分离到足够好的距离 用常规显微镜解决，从而有效分辨率约为70 nm。我们称这本小说 方法扩展显微镜（EXM）。从那以后，我们使技术更易于使用，创建了 EXM的版本我们称为Proexm（蛋白质保留EXM），该版本使用市售化学品 直接锚定为荧光团或抗体传播的荧光团锚固，可膨胀凝胶，然后 验证其在各种组织中保留纳米级特征的能力（自然界接受生物技术） 并将EXM扩展到RNA分子的锚定和膨胀，以互相纳米级 RNA可视化，我们称之为exfish（在自然方法上接受）。有很大的压抑需求 扩展3-D标本的纳米级成像方法，尤其是不需要专业的标本 设备;我们每周在麻省理工学院的小组中接待访客，以学习和练习EXM，并与 Janelia Research Campus我们将在2016年8月举办一个研讨会来教EXM动手。 为了使EXM解决神经科学中的许多问题，我们现在建议提高其功能和多功能性。 具体而言，我们（AIM 1）将为困难标本（例如秀丽隐杆线虫）开发优化的EXM形式，例如 以及单样本验证的策略（通过在样本中创建“物理规模杆”），（AIM 2）发明 在线性尺寸上将标本扩展到20倍或80倍的新化学物质，可实现〜15 nm和〜3 nm 分别有效的分辨率和（AIM 3）扩展EXM锚定化学家以可视化脂质 和DNA，以及生物分子的组合（例如，一次观察蛋白质，DNA和RNA）。我们的 项目将导致神经科学以及整个生物学具有极大适用性的工具。我们提出了一个 快节奏的4年技术开发赠款，该赠款将导致工具可实现大量 待分析的科学问题。我们将尽可能免费地分发所有工具，并进行其教学用法。