Protocol for neuron tracing and analysis of dendritic structures from noisy microscopy images using Neuronalyzer

Studying neuronal morphology requires imaging and accurate extraction of tree-like shapes from noisy microscopy data. Here, we present a protocol for automatic reconstruction of branched structures from microscopy images using Neuronalyzer software. We describe the steps for loading neuron images, denoising, segmentation, and tracing. We then detail feature extraction (e.g., branch curvature and junction angles), data analysis, and plotting. The software allows batch processing and statistical comparisons across datasets. Neuronalyzer is scale-free and handles noise and variation across images. For complete details on the use and execution of this protocol, please refer to Yuval et al.¹.