Tilted thick light-sheet microscopy with context-aware 3D localization for RNA imaging in intact 50 μm-thick sections

Neuron typing in intact brain sections demands 3D imaging reconciling thickness compatibility with large-area coverage, which is a persistent challenge for RNA imaging methods limited by either imaging area (< 1 mm²) or section thickness (< 20 μm). Here we introduce tilted thick light-sheet microscopy integrated with a context-aware 3D astigmatic localization algorithm based on continuous frame filtering, enabling multiplexed RNA imaging across 50 μm-thick tissue sections and centimeter-scale regions. The 45° illumination geometry achieves optical-interference-free imaging in a 50 μm-thick sample, while a context-aware 3D localization algorithm recovers submicron resolution by pinpointing fluorescence in situ hybridization (FISH) spots within volumetric excitation. This strategy could reveal layer-spanning neuronal distributions and region-specific expression gradients unattainable with conventional RNA imaging approaches. By resolving the trade-off between imaging depth and spatial coverage, our platform advances whole-brain transcriptomics and has the potential to carry out 3D neuron typing, which is critical for deciphering brain-wide cellular architectures.