Deep-tissue transcriptomics and subcellular imaging at high spatial resolution

Area of Science:

• Molecular Biology
• Microscopy Techniques
• Genomics

Background:

• Traditional fluorescence microscopy is limited by the number of available color channels, restricting simultaneous spatial analysis of multiple targets.
• Analyzing spatial organization of RNA and proteins in complex biological systems requires advanced imaging methods.

Purpose of the Study:

• To introduce and validate cycle hybridization chain reaction (cycleHCR) as a novel method for highly multiplexed imaging of RNA and proteins.
• To demonstrate the capability of cycleHCR for 3D spatial analysis in whole embryos and subcellular structures.

Main Methods:

• Developed cycleHCR by integrating multicycle DNA barcoding with hybridization chain reaction (HCR).
• Applied cycleHCR to whole-embryo transcriptomics imaging and combined it with expansion microscopy for subcellular resolution.
• Utilized cycleHCR for multiplex RNA and protein imaging in mouse hippocampal slices.

Main Results:

• Achieved precise 3D gene expression and cell fate mapping in whole embryos (~310 microm depth).
• Revealed intricate subcellular structures (10 types) in mouse embryonic fibroblasts using cycleHCR and expansion microscopy.
• Uncovered complex gene expression gradients and cell-type-specific nuclear variations in mouse hippocampal slices.

Conclusions:

• cycleHCR provides a versatile and quantitative framework for elucidating spatial regulation in deep tissues.
• This method overcomes limitations of traditional fluorescence microscopy for multiplexed imaging.
• cycleHCR has significant potential for biological research and diagnostic applications.