SkNeXt enables topology-guided neuronal reconstruction from petabyte-scale microscopy data

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.

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.

Volume microscopy, including electron and light microscopy, suffers from severe anisotropic resolution due to physical axial sectioning. Existing self-supervised axial super-resolution (ASR) methods face a trilemma bounded by overly smoothed regression textures, structural hallucinations of pure diffusion models, and prohibitive inference latency. In this paper, we propose Skeleton-refinE Microscopy (SkelEM), a self-supervised framework that decouples ASR at the training-signal level: a frozen topological network and a diffusion refiner are optimized by disjoint objectives, separating low-frequency topology formulation from high-frequency detail enhancement. Building on this deterministic skeleton, we exploit a unified cycle-consistent mechanism on input sparse slices to simultaneously extract a real-domain residual prior and bidirectionally align the diffusion refiner, washing away cross-plane artifacts without synthetic bias. By truncating the reverse diffusion process with this physical prior, SkelEM achieves high-fidelity detail restoration in merely $\le 5$ steps. To rigorously assess cross-instrument generalization, we further introduce BRAVE-ASR, a new benchmark of co-aligned anisotropic and isotropic volumes acquired on a Plasma-FIB instrument. Across public benchmarks, SkelEM achieves the most favorable balance across the fidelity-perception trade-off among self-supervised methods, with state-of-the-art downstream membrane segmentation performance and robust zero-shot generalization across distinct modalities.