Oblique Thick Light-Sheet Microscopy with Spatiotemporal 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—a persistent challenge for RNA imaging methods limited by either restricted fields-of-view (<1 mm ² ) or thin-section constraints (<20 μm). Here we introduce oblique thick light-sheet microscopy integrated with spatiotemporal 3D localization, enabling multiplexed RNA imaging across 50 μm-thick tissue sections and centimeter-scale regions. The 45° illumination geometry achieves optical-interference-free imaging in >100 μm-thick tissues during single-plane scans, while a spatiotemporal localization algorithm recovers submicron resolution by pinpointing fluorescence in situ hybridization (FISH) spots within volumetric excitation. This strategy reveals layer-spanning neuronal distributions and region-specific expression gradients unattainable with conventional RNA imaging approaches. By resolving the historical trade-off between imaging depth and spatial coverage, our platform advances whole-brain transcriptomics, enabling 3D neuron typing capability critical for deciphering brain-wide cellular architectures.