Three-color high-definition whole-brain imaging: simultaneous analysis of multiple anatomical structures with colocalized cytoarchitectural information.

Simultaneous multicolor whole-brain imaging with cytoarchitectural reference is critical for neural circuit mapping, but sequential scanning prolongs acquisition and complicates inter-channel registration. We developed a three-color high-definition fluorescent micro-optical sectioning tomography (HD-fMOST) system for three-channel high-throughput imaging of the whole mouse brain. We corrected chromatic aberration in the 405 nm illumination path and designed a precision 6-axis stage for pixel-level registration of three channels. Real-time DAPI counterstaining provides cytoarchitectural landmarks while preserving green/red fluorescence excitation. The system achieves sub-micron voxel resolution (0.32 × 0.32 × 1 μm3) and a high signal-to-background ratio. Validation on dual-color-labeled mouse brain demonstrates high-precision whole-brain imaging, accurate structural colocalization, and quantitative single-neuron morphological reconstruction.