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 w...