SKOOTS: Skeleton oriented object segmentation for mitochondria

Segmenting individual instances of mitochondria from imaging datasets can provide rich quantitative information, but is prohibitively time-consuming when done manually, prompting interest in the development of automated algorithms using deep neural networks. Existing solutions for various segmentation tasks are optimized for either: high-resolution three-dimensional imaging, relying on well-defined object boundaries (e.g., whole neuron segmentation in volumetric electron microscopy datasets); or low-resolution two-dimensional imaging, boundary-invariant but poorly suited to large 3D objects (e.g., whole-cell segmentation of light microscopy images). Mitochondria in whole-cell 3D electron microscopy datasets often lie in the middle ground--large, yet with ambiguous borders, challenging current segmentation tools. To address this, we developed skeleton-oriented object segmentation (SKOOTS)--a novel approach that efficiently segments large, densely packed mitochondria. SKOOTS accurately and efficiently segments mitochondria in previously difficult contexts and can also be applied to segment other objects in 3D light microscopy datasets. This approach bridges a critical gap between existing segmentation approaches, improving the utility of automated analysis of three-dimensional biomedical imaging data. We demonstrate the utility of SKOOTS by applying it to segment over 15,000 cochlear hair cell mitochondria across experimental conditions in under 2 hours on a consumer-grade PC, enabling downstream morphological analysis that revealed subtle structural changes following aminoglycoside exposure--differences not detectable using analysis approaches currently used in the field.