Accurate 3D neuron segmentation in fluorescence microscopy is critical for neuroscience. However, the sparse and elongated morphology of neurons poses significant challenges to existing segmentation methods. These methods struggle to preserve both local details and global topology, leading to fragmented results. To address this, we propose NeuroRefiner, a multi-agent system that formalizes the human expert workflow involving iterative global observation and local editing. Specifically, NeuroRefiner comprises three collaborative agents dedicated to diagnosing topological errors, generating correction instructions, and validating refinement quality. To facilitate agent instruction-guided segmentation refinement, we propose TopoRefineNet, a dedicated 3D U-Net-based tool that leverages cross-modality feature fusion to generate refined masks. Through multi-round agent reasoning and voxel-level editing, NeuroRefiner produces topologically more accurate segmentations with enhanced interpretability. Experiments on the BigNeuron, CWMBS, and ZBFWB datasets demonstrate that NeuroRefiner outperforms state-of-the-art methods, notably achieving a 3.02% improvement in F1 score on the challenging ZBFWB dataset.
Neuron counting and segmentation in microscopy images of neuronal cultures is a routine and time-consuming task in neuroscience research, traditionally performed through manual inspection or semi-automatic tools. We present NeuroAdaptTrainer, an open-source Fiji/ImageJ plugin that integrates a YOLO instance-segmentation model directly into the microscopist's workflow. The plugin allows a user to run automatic neuron detection on a single image or a batch of images, manually correct the resulting detections from within Fiji, and use those corrections to adapt the model to new imaging conditions via transfer learning. A built-in external validation module allows the base and adapted models to be compared quantitatively on a held-out annotated set. NeuroAdaptTrainer lowers the barrier for non-specialist users to benefit from deep-learning-based segmentation while keeping expert supervision at the center of the workflow.
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.