PROJECT SUMMARY Synapses are intricate structures smaller than 1 micron, encompassing thousands of subtypes, each defined by a unique composition of functional proteins. The ability to map the 3D organization of these proteins is key for comprehending the molecular anatomy and mechanisms underlying brain functions and diseases. Conventional optical microscopy, limited by a resolution of ~250 nm, falls short of resolving the complex spatial organization of synaptic proteins. Meanwhile, electron microscopy (EM) and various super- resolution optical imaging techniques like STED and SMLM are hindered by a lack of molecular specificity, challenges in 3D imaging, and the labor-intensive, slow, and costly nature of the operation. This gap in technology has left synapse typing unaddressed until the recent development of the next-generation Expansion Microscopy technique, Magnify. Magnify offers an ~11-fold linear expansion of biological specimens in a single round, ensuring efficient retention of proteins, nucleic acids, and lipids. It is compatible with various tissue types and fixation methods while preserving epitopes for high-plex imaging. By reaching ~15 nm resolution, Magnify can discern ultrastructural details such as cilia, mitochondria cristae, and synaptic densities. We seek to build the prototype of the Multiplex Magnify sample preparation kits as a universal tool for synapse typing and brain imaging. Our goal is to equip neuroscientists with the ability to perform EM-quality 3D ultrastructural analysis, mapping an extensive array of biomolecular markers within the intricate synaptic structures in brain tissue at a fraction of the cost and time using only a light microscope. In our project, we will enhance the capabilities of our Magnify technology in neuroscience research through the following Aims: (1) validate Magnify-compatible antibodies and a novel direct immunostaining strategy for key synaptic markers to generate a library of labels for neuroscience researchers; (2) develop a mounting strategy for multiplex immunofluorescence imaging of synapses to ensure high-fidelity images through at least 10 rounds of imaging; and (3) develop a multiplex Magnify kit prototype for easy adoption for neuroscience research. Upon successful completion, Magnify Biosciences will produce Multiplex Magnify sample preparation kits for neuroscientists conducting simultaneous 3D ultrastructural analysis, neuronal tracing, synapse typing, etc., in brain tissue. Our work will prepare for a Phase II grant to increase multiplexing with barcoded oligo-conjugated Flexible binders and a larger antibody panel and to advance the prototype with an automated Auto-Magnify device for autonomous, multiplexed nanoscale imaging, enhancing super-resolution imaging accessibility for advanced brain research.