This work provides an empirical reliability assessment of three‑dimensional mitochondrial morphometry pipelines. Using 2,720 development objects from the 3D Mitochondria Shape Library, occupancy‑derived volumes exceed reference mesh volumes by an average of 3.665% while the intraclass correlation coefficient remains 0.994. Boundary analysis reveals an outward label displacement of 0.00304 normalized units. In a controlled label‑pipeline reimplementation, removing the depth offset reduces volume error across 55 analyzed objects by a mean of 1.57 percentage points (≈45% of the total inflation); the remaining error source is not isolated. A frozen regression model built on occupancy features lowers the median absolute percentage error from 3.481% to 0.664% on 2,728 previously unused objects, yet its calibrated error bound covers only 92.1% overall and 49.2% in a low‑occupancy subgroup, indicating that accuracy and uncertainty transfer must be evaluated separately. In 550 rat‑cortex objects from the MitoEM resource, coarsening in‑plane spacing from 8 nm to 24 nm changes median surface area by –10.60% and sphericity by +11.76%, despite a rank correlation of 0.994. These findings supply quantitative checks to separate processing‑induced descriptor changes from genuine biological differences, without asserting biological invariance or cross‑source correction transfer.
Review: The study convincingly demonstrates that imaging pipelines introduce systematic yet subtle biases in mitochondrial volume and shape metrics, underscoring the necessity of dedicated correction procedures and uncertainty quantification when comparing results across datasets.