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使分子生物学成为可能的技术 在整个保存的 3-D 过程中成像的信息（例如，特定核酸和蛋白质的身份） 因此，我们开发了一种全新的方法。 2015 年《科学》杂志上发表的方法：与早期光学显微镜的放大方法相比， 它依靠镜头光学放大细胞和组织的图像，我们物理放大保存的 通过直接在样品内机械合成可膨胀的聚电解质凝胶。 将样本均质化，然后在水中透析，我们可以将组织的线性尺寸扩大约 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远的距离 可以用传统显微镜解决，从而产生约 70 nm 的有效分辨率，我们称之为新颖。 从那时起，我们使该技术变得更易于使用，创建了一种方法。 ExM 的版本，我们称之为 proExM（蛋白质保留 ExM），它使用市售化学品来 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，并且 验证其在各种组织中保留纳米级特征的能力（被 Nature Biotechnology 接受） 并将 ExM 扩展到纳米级 RNA 分子的锚定和扩展 RNA 可视化，我们称之为 ExFISH（被 NatureMethods 接受）。 用于扩展 3D 样本的纳米级成像方法，特别是不需要专门的方法 设备；我们每周在麻省理工学院的小组中接待访客来学习和练习 ExM，并与 我们将在 2016 年 8 月在 Janelia 研究园区举办一个研讨会，教授 ExM 实践操作。鉴于潜力 为了让 ExM 解决神经科学中的许多问题，我们现在建议增强其功能和多功能性。 具体来说，我们将（目标 1）为困难样本（例如秀丽隐杆线虫）开发 ExM 的优化形式，如 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 新的化学物质可将样本的线性尺寸扩大 20 倍或 80 倍，实现 ~15 nm 和 ~3 nm 分别有效的分辨率，以及（目标 3）扩展 ExM 锚定化学以实现脂质的可视化 和 DNA，以及生物分子的组合（例如，同时看到蛋白质、DNA 和 RNA）。 该项目将产生在神经科学以及整个生物学领域具有广泛适用性的工具。 快节奏的、为期 4 年的技术开发补助金，将产生能够实现大量 我们将尽可能免费地分发所有工具，并教授其使用方法。