Abstract Neural circuits composed of interconnected neurons with distinct properties lay the physical foundation of any brain function. Identifying connections between individual neurons is central to understand how information is processed and propagated in the brain. While emerging high throughput light microscopy technologies are highly promising in allowing whole brain scale imaging at the single cell level, optical resolution limitation prevents their use in differentiating densely labeled neuronal processes in the same brain. In addition, computational tools for automatically extracting morphological information from intermingled neurons with high accuracy are still lacking. Our team will concurrently develop novel genetic tools for neuronal labeling, super-resolution imaging, and automated neuronal tracing for high-throughput circuit reconstruction. We will apply these tools to obtain densely reconstructed inhibitory microcircuits in the mouse cortex. These tools will be readily applicable for studying other long-standing questions in neuroscience and the resources generated by this project will be useful for future computational tool development.