Penumbria: Advanced 3D cell segmentation for biomedical imaging

Quantitative analysis of three-dimensional cellular architecture is fundamental to understanding tissue organization, disease progression, and drug response. There are effective approaches for 2D segmentation, yet 3D cell segmentation remains a critical bottleneck due to diverse cell morphologies, low signal-to-noise ratios, and data scarcity. We introduce Penumbria, a general-purpose 3D cell segmentation framework that achieves state-of-the-art accuracy across morphologically distinct cell populations and imaging conditions in volumetric microscopy. Penumbria formulates segmentation as a regression problem on distances to cell boundaries, supporting instance reconstruction without shape priors and permitting end-to-end GPU inference. A U-Net-based architecture with xLSTM bottleneck blocks and patch embeddings enables multi-scale feature extraction, long-range modeling of spatial context, and convolutional feature-volume tokenization. The model is extended with two modules: a Global Zernike Phase Layer, which learns Zernike-parameterized phase corrections in the frequency domain to deal with optical aberrations such as defocus and tilt, and a Scaled Geocaps Layer, which samples features at fixed grid locations across multiple spatial scales, routing evidence between them such that a detection is only confident where concordance holds across scales simultaneously. Across four diverse 3D datasets selected to probe the limits of existing methods, Penumbria outperforms Cellpose-SAM on all four and StarDist-3D on three, with comparable accuracy on the fourth, achieving up to a 38% improvement in mean average precision over the second-best method. Penumbria’s strong boundary accuracy further supports downstream analyses such as quantifying membrane dynamics or protein localization. We also release a hand-labeled confocal dataset of 1,543 zebrafish neurons across seven volumes, on which Penumbria can be easily and quickly trained.