From Manual to Macro: A Reproducible Fiji Workflow for Semi-automated Collagen Fibril Diameter Quantification in Transmission Electron Microscopy.

Quantitative assessment of collagen fibril diameter is essential for understanding ultrastructural changes in aging, connective tissue disorders, and extracellular matrix remodeling. Although transmission electron microscopy (TEM) is widely used for this purpose, existing methods for fibril measurement are predominantly manual, operator-dependent, and prone to inconsistent reporting. To address this, we developed a semi-automated Fiji/ImageJ (IJ1) macro that standardizes fibril diameter measurements from two-dimensional TEMs. The macro uses a wand-based region-of-interest (ROI) detection strategy with integrated geometric validation. It calculates multiple metrics, including area-equivalent diameters, ellipse-derived major and minor axes, shape consistency indices, and spatial localization across the image field. Built-in quality assurance thresholds exclude oblique or irregular profiles, ensuring accurate identification of true fibril cross-sections. Real-time visual overlays support live validation and user feedback during analysis. We detail the implementation, analytical workflow, and validation approach, provide practical guidance for reproducible use across operators and datasets, and show proof-of-concept utility for analysis of collagen fibrils in vascular Ehlers-Danlos syndrome (VEDS) subject dermal samples. This open-source, extensible tool enhances standardization and reproducibility in collagen fibril morphometry for ultrastructural research.