Living Additive Expansion Microscopy

项目总结/摘要 膨胀显微镜（ExM）是一种功能强大的新成像技术，它可以物理放大组织样本， 在传统显微镜上实现超分辨率成像。膨胀过程依赖于 以及生物样本内的生物网络的扩展。有效解决方案已完成 由ExM直接相关的扩展实现的因素（有效分辨率=（原始 分辨率）/（扩展因子））。典型的程序将样品扩大到4- 4.5倍的原始尺寸， 将传统光学显微镜的分辨率从~ 300 nm提高到~ 70 nm。更高的有效分辨率 因此，可以通过增加膨胀来实现;然而，膨胀过程最终受到以下因素的限制 网络溶胀的热力学和凝胶的静态性质，其中聚合物链是“死的”， 在聚合后不能进一步生长。活体增材制造（LAM）是一种新的合成方式 不受膨胀极限限制的聚合物凝胶。LAM依赖于光控制的自由基聚合， 在每个网络链中具有嵌入的光活性三硫代碳酸酯（TTC）基团的聚合物网络。在 在单体和光的存在下，网络链的聚合被引发，消耗单体并增长， 聚合物网络在每个方向上相等。由于LAM使用可控聚合， 终止被最小化，从而能够在光照射下重新引发和连续生长网络， 基本上不限制可实现的增长/扩展因素。在本提案中，我们的目标是将联合收割机 和ExM，以实现前所未有的扩张水平，我们称之为“生活添加剂扩张”。 显微镜检查（LAExM）。TTC-凝胶合成和光生长将针对生物组织进行优化， 以各向同性方式生长嵌入式网络。预计LAExM将实现近- 组织网络的无限扩张，从而消除了由于可接近扩张而导致的任何电流限制 ExM中的因素。因此，LAExM将用于获得重要的 与记忆和学习相关的超分子结构，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。这项提议需要约翰逊夫妇之间的广泛合作， Boyden和Tsai小组，以及麻省理工学院的显微镜和成像设备。培训将由 约翰逊和博伊登实验室的成员优化化学和组织生长方案， 分别Tsai研究小组将为以下相关组织中淀粉样斑块的成像提供指导： 老年痴呆症每月举行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的科学环境和约翰逊，博伊登和蔡 实验室，所有这些都促进了跨几个部门的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。

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.