Living Additive Expansion Microscopy

活性添加剂膨胀显微镜

• 批准号: 10271264
• 负责人: Megan Hill
• 金额: $ 6.18万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-30 至 2022-08-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10271264
• 关键词: 3-Dimensional; Actins; Alzheimer' s Disease; Antibodies; Axon; Biological; Chemicals; Chemistry; Collaborations; Complex; Consumption; Core Facility; Development; Disease; Ensure; Environment; Faculty; Gel; Growth; Image; Imaging Techniques; Label; Learning; Length; Light; Memory; Methods; Microscope; Microscopy; Microtubules; Modernization; Molecular; Mus; Nature; Neurons; Optics; Parents; Polymers; Procedures; Process; Protocols documentation; Research; Resolution; Sampling; Senile Plaques; Specimen; Spectrin; Stains; Structure; Swelling; Techniques; Technology; Thermodynamics; Three-Dimensional Image; Time; Tissue Embedding; Tissue Expansion; Tissue Sample; Tissues; Training; Water; Work; amyloid imaging; base; biological systems; brain parenchyma; brain tissue; crosslink; density; imaging facilities; instrumentation; irradiation; meetings; member; microscopic imaging; monomer; nanoscale; operation; polymerization; restraint; tool; trithiocarbonate

PROJECT SUMMARY/ABSTRACT Expansion microscopy (ExM) is a powerful new imaging technique that physically magnifies tissue samples to enable super-resolution imaging on conventional microscopes. The expansion process relies on the synthesis and expansion of a polyelectrolyte network within a biological specimen. The effective resolution accomplished by ExM is directly related to the factor of expansion achieved (effective resolution = (original resolution)/(expansion factor)). Typical procedures expand samples to 4–4.5x their original size, thereby enhancing resolution on conventional optical microscopes from ~300nm to ~70nm. Greater effective resolution can therefore be achieved with increasing expansion; however, the expansion process is ultimately limited by the thermodynamics of network swelling and the static nature of the gel, in which the polymer chains are “dead”, unable to grow further after the polymerization. Living Additive Manufacturing (LAM) is a new way to synthesize polymer gels unconfined by the limits of expansion. LAM relies on the photocontrolled radical polymerization of a polymer network with embedded photoactive trithiocarbonate (TTC) groups in each network strand. In the presence of monomer and light, polymerization of network strands is initiated, consuming monomer and growing the polymer network equivalently in each direction. Because LAM uses controlled polymerization, chain termination is minimized, thereby enabling reinitiation and continual growth of the network under light irradiation, with essentially no restraints on achievable growth/expansion factors. In this proposal, we aim to combine LAM and ExM to achieve unprecedented levels of expansion in a process we call Living Additive Expansion Microscopy (LAExM). TTC-gel synthesis and photogrowth will be optimized for biological tissue and the ability to grow the embedded network in an isotropic manner will be analyzed. LAExM is anticipated to enable near- limitless expansion of the tissue network, thereby removing any current limitations due to accessible expansion factors in ExM. LAExM will therefore be employed to obtain ultrahigh resolution images of important supramolecular structures associated with memory and learning such as actin and spectrin in neurons and amyloid plaques in brain parenchyma. This proposal requires extensive collaboration between the Johnson, Boyden, and Tsai groups, in addition to the microscopy and imaging facilities at MIT. Training will be done by members of the Johnson and Boyden labs for the optimization of the chemistry and tissue growth protocols, respectively. The Tsai group will provide guidance in the imaging of amyloid plaques in tissue associated with Alzheimer’s disease. Monthly meetings will be held to evaluate results and assess or optimize the current training plan. The proposed work will benefit from the scientific environment at MIT and the Johnson, Boyden and Tsai labs, all of which promote co-operation and collaboration with knowledgeable faculty across several departments and provide access to the necessary instrumentation and core facilities.

项目摘要/摘要 扩展显微镜（EXM）是一种强大的新成像技术 在常规显微镜上启用超分辨率成像。扩展过程依赖于合成 以及在生物标本内的聚电解质网络的扩展。有效的分辨率完成了 由EXM与实现的扩展因素直接相关（有效分辨率=（原始） 分辨率）/（扩展因子））。典型的程序将样本扩展到4-4.5倍，因此 从〜300nm到〜70nm上对常规光学显微镜的分辨率提高分辨率。更大的有效分辨率 因此，可以通过增加的扩展来实现；但是，扩展过程最终受到 网络肿胀的热力学和凝胶的静态性质，其中聚合物链“死”， 聚合后无法进一步生长。生活增材制造（LAM）是合成的新方法 聚合物凝胶因膨胀限制而不受限制。 LAM依赖于光控制的自由基聚合 每个网络链中具有嵌入式光活性三氧碳酸盐（TTC）组的聚合物网络。在 单体的存在和光的存在，启动网络链的聚合，消耗单体并增长 聚合物网络在每个方向上平均。由于LAM使用受控聚合，因此 终止是最小化的，从而使网络在光照射下对网络的持续增长， 基本上没有对可实现的增长/扩展因素的限制。在此提案中，我们的目标是结合Lam 和EXM在我们称为“生活添加剂”的过程中实现前所未有的扩展水平 显微镜（LAEXM）。 TTC-GEL合成和光流将针对生物组织进行优化和能力 要以各向同性方式发展嵌入式网络。预计Laexm将使接近 - 组织网络的无限扩展，从而消除了由于可及扩展而引起的任何当前局限性 EXM中的因素。因此，LaExm将被用来获得重要的重要分辨率图像 与记忆和学习相关的超分子结构，例如神经元中的肌动蛋白和谱蛋白，以及 大脑实质中的淀粉样斑块。该建议需要约翰逊之间的广泛合作 博伊登（Boyden）和泰（Tsai）组除了在麻省理工学院（MIT）的显微镜和成像设施外。培训将由 约翰逊和博伊登实验室的成员，以优化化学和组织生长方案， 分别。 TSAI组将提供与组织中淀粉样蛋白斑块成像的指导 阿尔茨海默氏病。将举行每月会议以评估结果并评估或优化当前的培训 计划。拟议的工作将受益于麻省理工学院和约翰逊，博伊登和泰的科学环境 实验室，所有这些都促进了与多个部门知识渊博的教师的合作与合作 并提供对必要的仪器和核心设施的访问。