SkeleSketch: An Open-Source Tool for Quantifying Microglial Morphology in Fiji/ImageJ
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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.
Significance Statement
Quantitative analysis of microglial morphology is increasingly used to study how these cells respond to injury, disease, and aging. However, in practice, it remains difficult for many laboratories. Manual tracing is labor-intensive and subjective, whereas automated pipelines typically require high-resolution imaging, expensive software, or computational expertise. SkeleSketch was developed to fill the gap between these approaches. It is a free, open-source Fiji/ImageJ tool that operates on standard epifluorescence images and requires only modest computing resources and minimal technical expertise, with optional user correction where automated tracing struggles. By lowering the technical and financial barriers to reproducible morphometric analysis, SkeleSketch broadens access to quantitative, single-cell microglial phenotyping across basic, translational, and educational neuroscience.
