Stereology with OPEN-Stereo: low-cost, accessible, and accurate cellular quantification

Critical to rigor in neurodegeneration research is the accurate and unbiased assessment of degenerative phenotypes, where stereology remains the gold standard, yet its widespread adoption is hindered by the high cost of proprietary systems. We developed OPEN-Stereo, an open-source stereology platform that integrates standard microscopy hardware with intelligent, software-based calibration and positional control. Innovative use of computer vision methods, multi-scale image-based calibration and navigation, are combined with open-loop stage data to establish a global positioning system without costly hardware, while remaining within the accuracy tolerances inherent to stereological sampling. OPEN-Stereo implements the stereological random sampling method for unbiased cell counting. Validation using samples previously analyzed on commercial systems demonstrated up to 95% agreement in cell counts across multiple brain regions, with statistical equivalence confirmed by two-way repeated measures ANOVA (p = 0.8962). Beyond enabling accessibility, OPEN-Stereo's image-analytic architecture enables stereology to evolve beyond manual practice, toward AI-driven cell identification and counting.