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

Area of Science:

• Neuroscience
• Microscopy
• Molecular Biology

Background:

• Accurate neuron typing requires 3D imaging of intact brain sections.
• Existing RNA imaging methods face limitations in balancing imaging area and section thickness.
• Challenges persist in achieving high-resolution, large-area RNA imaging in thick tissues.

Purpose of the Study:

• To develop an advanced RNA imaging method for 3D neuron typing in thick brain sections.
• To overcome the limitations of conventional RNA imaging techniques regarding area coverage and section thickness.
• To enable high-resolution, large-scale transcriptomic analysis in the brain.

Main Methods:

• Introduced tilted thick light-sheet microscopy with a 45° illumination geometry.
• Integrated a context-aware 3D astigmatic localization algorithm with continuous frame filtering.
• Enabled multiplexed RNA imaging in 50 μm-thick tissue sections across centimeter-scale regions.

Main Results:

• Achieved optical-interference-free imaging in 50 μm-thick samples.
• Recovered submicron resolution by pinpointing fluorescence in situ hybridization (FISH) spots within volumetric excitation.
• Demonstrated the capability for layer-spanning neuronal distribution and region-specific expression gradient analysis.

Conclusions:

• The developed platform resolves the trade-off between imaging depth and spatial coverage for RNA imaging.
• Advances whole-brain transcriptomics and 3D neuron typing capabilities.
• Provides a novel approach for deciphering brain-wide cellular architectures.