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Background and Objective: Advances in electron microscopy (EM) now allowthree-dimensional (3D) imaging of hundreds of micrometers of tissue withnanometer-scale resolution, providing new opportunities to study theultrastructure of the brain. In this work, we introduce a freely availableMatlab-based gACSON software for visualization, segmentation, assessment, andmorphology analysis of myelinated axons in 3D-EM volumes of brain tissuesamples. Methods: The software is equipped with a graphical user interface(GUI). It automatically segments the intra-axonal space of myelinated axons andtheir corresponding myelin sheaths and allows manual segmentation,proofreading, and interactive correction of the segmented components. gACSONanalyzes the morphology of myelinated axons, such as axonal diameter, axonaleccentricity, myelin thickness, or g-ratio. Results: We illustrate the use ofthe software by segmenting and analyzing myelinated axons in six 3D-EM volumesof rat somatosensory cortex after sham surgery or traumatic brain injury (TBI).Our results suggest that the equivalent diameter of myelinated axons insomatosensory cortex was decreased in TBI animals five months after the injury.Conclusions: Our results indicate that gACSON is a valuable tool forvisualization, segmentation, assessment, and morphology analysis of myelinatedaxons in 3D-EM volumes. It is freely available athttps://github.com/AndreaBehan/g-ACSON under the MIT license.

gACSON software for automated segmentation and morphology analyses of myelinated axons in 3D electron microscopy