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.

项目概要/摘要 膨胀显微镜 (ExM) 是一种强大的新型成像技术，可物理放大组织样本 在传统显微镜上实现超分辨率成像。扩展过程依赖于合成 以及生物样本内聚电解质网络的扩展。已达成有效决议 ExM 与所实现的扩展系数直接相关（有效分辨率=（原始 分辨率）/（扩展因子））。典型程序将样本扩大到原始大小的 4–4.5 倍，从而 将传统光学显微镜的分辨率从 ~300nm 提高到 ~70nm。更有效的分辨率 因此可以通过增加扩展来实现；然而，扩张过程最终受到以下因素的限制： 网络膨胀的热力学和凝胶的静态性质，其中聚合物链是“死的”， 聚合后无法进一步生长。活性增材制造（LAM）是一种新的合成方法 聚合物凝胶不受膨胀限制。 LAM 依赖于光控自由基聚合 每个网络链中嵌入光活性三硫代碳酸酯（TTC）基团的聚合物网络。在 当单体和光存在时，网络链开始聚合，消耗单体并生长 聚合物网络在每个方向上等效。由于 LAM 使用受控聚合，链 终止被最小化，从而使得网络在光照射下能够重新启动和持续生长， 对可实现的增长/扩张因素基本上没有限制。在这个提案中，我们的目标是结合 LAM 和 ExM 在我们称为“活性添加剂扩张”的过程中实现前所未有的扩张水平 显微镜（LAExM）。 TTC-凝胶合成和光生长将针对生物组织和能力进行优化 将分析以各向同性方式生长嵌入式网络。 LAExM 预计将实现近 组织网络的无限扩展，从而消除了由于可扩展性而导致的任何当前限制 ExM 中的因素。因此，LAExM 将用于获取重要的超高分辨率图像 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血影蛋白 脑实质中的淀粉样斑块。该提案需要约翰逊、 Boyden 和 Tsai 小组，以及麻省理工学院的显微镜和成像设施。培训将由 Johnson 和 Boyden 实验室的成员负责优化化学和组织生长方案， 分别。 Tsai 小组将为与以下疾病相关的组织中淀粉样斑块的成像提供指导： 阿尔茨海默病。将每月举行一次会议来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以及 Johnson、Boyden 和 Tsai 的科学环境 实验室，所有这些都促进与多个部门知识渊博的教师的合作与协作 并提供必要的仪器和核心设施。