ABSTRACT
Progress in histological methods and in microscope technology has enabled dense staining and imaging of axons over large brain volumes, but tracing axons over such volumes requires new computational tools for 3D reconstruction of data acquired from serial sections. We have developed a computational pipeline for automated tracing and volume assembly of densely stained axons imaged over serial sections, which leverages machine learning-based segmentation to enable stitching and alignment with the axon traces themselves. We validated this segmentation-driven approach to volume assembly and alignment of individual axons over centimeter-scale serial sections and show the application of the output traces for analysis of local orientation and for proofreading over aligned volumes. The pipeline is scalable, and combined with recent advances in experimental approaches, should enable new studies of mesoscale connectivity and function over the whole human brain.