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Multiplexed And High-throughput Neuronal Fluorescence Imaging With Diffusible Probes

Syuan-Ming Guo, Remi Veneziano, Simon Gordonov, Li Li, Eric Danielson, Karen Perez de Arce, Demian Park, Anthony B. Kulesa, Eike-Christian Wamhoff, Paul C. Blainey, Edward S. Boyden, Jeffrey R. Cottrell, Mark Bathe

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Abstract Synapses contain hundreds of distinct proteins whose heterogeneous expression levels are determinants of synaptic plasticity and signal transmission relevant to a range of diseases. Here, we use diffusible nucleic acid imaging probes to profile neuronal synapses using multiplexed confocal and super-resolution microscopy. Confocal imaging is performed using high-affinity locked nucleic acid imaging probes that stably yet reversibly bind to oligonucleotides conjugated to antibodies and peptides. Super-resolution PAINT imaging of the same targets is performed using low-affinity DNA imaging probes to resolve nanometer-scale synaptic protein organization across nine distinct protein targets. Our approach enables the quantitative analysis of thousands of synapses in neuronal culture to identify putative synaptic sub-types and co-localization patterns from one dozen proteins. Application to characterize synaptic reorganization following neuronal activity blockade reveals coordinated upregulation of the post-synaptic proteins PSD-95, SHANK3 and Homer-1b/c, as well as increased correlation between synaptic markers in the active and synaptic vesicle zones.