Abstract
Reconstructing single-neuron projectomes is essential for mapping the mesoscopic connectome and eventually for understanding brain-wide connectivity and diverse brain functions. The combination of sparse labeling techniques and large-scale and high-resolution optical imaging technologies has been revolutionizing the brain-wide reconstruction of single-neuron morphologies, as exemplified by the dataset for over 10,100 single-neuron projectomes of hippocampal neurons. Here, we illustrate a comprehensive protocol for large-scale single-neuron reconstruction in the mouse brain. This includes key steps and examples in imaging data preprocessing, neurite tracing, and registration into a template brain. These procedures enable efficient and accurate large-scale morphological reconstruction of single neurons in the mouse brain. Key features • Rigorous and effective single-neuron reconstruction from raw imaging data. • Multi-person tracing and quality control for the accuracy of single-neuron tracing results. • Precise image registration based on landmark drawings of selected brain regions.