Catena: A Comprehensive Software Suite for Large-Scale Connectomics

Proofreading--correcting segmentation errors in 3D brain reconstructions--is the rate-limiting step in synapse-resolution connectomics. We release ConnectomeBench2, a unified multi-species dataset of over 716,485 expert-labeled proofreading decisions with >4,500,000 associated images spanning four major open connectomes (mouse, human, zebrafish, fly), spanning both split and merge error correction. Trained on this dataset, a single Vision Transformer with shared encoders for mesh geometry and electron microscopy reaches human-level accuracy across species for split error correction and merge error identification, with performance scaling with data size and modality. Beyond accuracy, we show that the model is well-calibrated within distribution, that measures of distribution distance predict where calibration and accuracy will degrade on unseen data, and that connectomics-specific pretraining and active learning-based sample selection show potential to substantially reduce the labeling effort needed to extend to new species and brain regions. The benchmark provides the infrastructure to train and evaluate increasingly capable vision models for connectomic proofreading. Data and code availability. The ConnectomeBench2 dataset is released on Hugging Face at https://huggingface.co/datasets/jeffbbrown2/ConnectomeBench2. The accompanying codebase is available on GitHub at https://github.com/timfarkas/ConnectomeBench2.

Establishing large-scale, high-resolution neural connectivity maps is fundamental to elucidating the structural basis of brain function. However, when processing terabyte- or petabyte-scale electron microscopy data, over-segmentation inherent in automated reconstruction algorithms remains a critical bottleneck, requiring extensive manual proofreading spanning person-years. To alleviate the heavy reliance on annotated data and the limited flexibility of conventional tracing methods, we propose a training-free, targeted neuron tracing framework. Specifically, we introduce a skeleton-guided Heuristic Spatial Search paradigm that leverages geometric priors to iteratively reconstruct neuronal morphologies through a probing-verification cycle. To achieve robust zero-shot semantic verification, we further develop a Dimension-Aware Semantic Verification strategy built upon the foundation model NeuroSAM 2. This strategy resolves intra-slice splits via Planar Ensemble Consensus and inter-slice splits via Axial Spatio-Temporal Propagation. Notably, we integrate the proposed workflow into the Neuroglancer visualization platform, enabling an interactive human-in-the-loop proofreading system. Experimental results demonstrate that the proposed method outperforms supervised baselines and reduces manual proofreading time by 33.4%. The source code is publicly available at https://github.com/HeadLiuYun/Probe-EM.

Recent advances in high-resolution fluorescence and electron microscopy have enabled nanoscale imaging across increasingly large brain volumes, but the resulting terabyte- to petabyte-scale datasets make complete neuronal reconstruction prohibitively expensive in computation, data movement, and manual proofreading. Here, we present SkNeXt, a topology-first framework for scalable neuronal reconstruction from large volumetric microscopy datasets. Instead of densely processing entire image volumes, SkNeXt first converts neuronal morphology into compact SWC skeletons that preserve long-range connectivity. Proofreading is therefore focused on sparse neuronal trees, allowing branch, continuity, and connectivity errors to be corrected before high-resolution reconstruction. The corrected skeletons then serve as persistent structural priors for recovering detailed morphology while preserving neuronal identity and topology. Crucially, SkNeXt also uses neuronal skeletons as spatial indices for selective data access, retrieving high-resolution image regions only along reconstructed trajectories and bypassing most background and signal-free volumes. This substantially reduces I/O and computational overhead, allowing reconstruction cost to scale with neuronal morphology rather than total dataset size. Using SkNeXt, we reconstructed neurons from a petabyte-scale super-resolution fluorescence dataset of the mouse brain on a single GPU within one week, without requiring exhaustive dense inference across the complete imaging volume.