SkeleSketch: An Open-Source Tool for Quantifying Microglial Morphology in Fiji/ImageJ

Abstract

Microglial morphology is widely used as an indicator of cellular state, yet existing analytical approaches are often labor-intensive, require proprietary software, or depend on high-resolution imaging platforms. Here, SkeleSketch is presented as an open-source Fiji/ImageJ plugin that automates microglial skeletonization, morphological quantification, and data organization while allowing optional user-guided correction. Using spinal cord microglia from PBS- and LPS-treated mice, SkeleSketch accurately reconstructed cellular morphology and captured expected LPS-induced morphological remodeling, including enlarged soma size and reduced branching complexity. Comparable measurements were obtained from both CX3CR1-GFP reporter mice and Iba1 immunostaining, demonstrating robustness across labeling strategies. Additionally, SkeleSketch morphometrics were positively correlated with those generated by the commercial software Imaris. Our pipeline provides an accessible, reproducible, and flexible platform for quantitative microglial morphology analysis using standard epifluorescence microscopy.