Quantitative Multiplexed Super-Resolution Neuronal Synapse Imaging using DNA-Paint

Abstract

Neuronal synapses form critical junctions of communication in neuronal networks, mediating neuronal signal transmission and circuit function. Synapses consist of thousands of proteins organized on the sub-micron scale, and their dysregulation via genetic aberrations including copy number variations and site-specific mutations is associated with a large number of neurological and psychiatric diseases. Understanding how these genetic aberrations affect the localization and structural organization of synapse proteins at the single synapse level is crucial for understanding neuronal function and related pathogenesis. Super-resolution fluorescence imaging is a powerful approach to resolving nanometer-scale organization of synapse molecules. However, conventional super-resolution imaging is limited to simultaneous interrogation of only 2-4 proteins in a single synapse. As an alternative, here we apply DNA-PAINT (Points Accumulation for Imaging in Nanoscale Topography) that enables highly multiplexed super-resolution imaging of synaptic proteins. PAINT generally employs transiently binding imaging probes to molecular targets in order to generate target blinking while simultaneously allowing probe wash-out or exchange, thereby in principle enabling sequential imaging of arbitrary numbers of molecular targets using a single dye and laser source. Use of single-stranded DNA as the soluble fluorescent probe that targets complementary single-stranded DNAs on cognate antibodies facilitates arbitrary blinking events per spatial localization. We employ this approach to resolve the localization and organization of synaptic proteins simultaneously with cytoskeletal markers for microtubules and actin, and demonstrate how fluorescence correlation analysis can be used to quantify their copy number in a highly multiplexed manner at the single cell and synapse level.