Probe-EM: Targeted Neuron Tracing via Training-Free Semantic Verification

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.

Automatic neuron reconstruction from light microscopy images is a central problem in computational neuroanatomy. While recent methods have achieved encouraging results on local image blocks, it remains unclear whether such progress translates to reconstruction that is both structurally accurate and scalable to the whole-brain scale. We present CORAL, the first benchmark for structure-aware evaluation of automatic neuron reconstruction from light microscopy images at both local and whole-brain scales. Built on a high-quality whole-brain fMOST dataset with carefully curated annotations, CORAL establishes two progressive tasks: block-level reconstruction, which evaluates reconstruction methods under limited spatial context, and brain-wide reconstruction, which assesses complete neuron reconstruction at the whole-brain scale. To account for topological correctness beyond geometric distance similarity, we introduce a structure-aware metric based on fiber prediction. To further achieve complete neuron reconstruction across the entire brain, we develop a brain-wide neuron tracing framework that extends arbitrary local reconstruction methods to the whole-brain scale through an iterative local-to-global process. Using this benchmark, we provide the first structure-aware comparison of mainstream methods for local neuron reconstruction and further evaluate their performance in brain-wide reconstruction. Our results underscore the importance of structure-aware evaluation and the need for more robust methods for complete neuron reconstruction.

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.