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Excitation-Scanning Hyperspectral Imaging Microscopy to Efficiently Discriminate Fluorescence Signals
Published on: August 22, 2019
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HCR spectral imaging: 10-plex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent
Biorxiv : the Preprint Server for Biology
|September 11, 2023
Summary
Hybridization chain reaction (HCR) spectral imaging enables simultaneous visualization of 10 RNA and protein targets in challenging samples. This advanced technique provides quantitative, high-resolution imaging in whole-mount embryos and brain sections.
Area of Science:
- Molecular Biology
- Biophysics
- Imaging Science
Background:
- Multiplex imaging of RNA and proteins is crucial for understanding biological processes.
- Conventional imaging methods struggle with spectral overlap and autofluorescence in complex samples.
- Existing techniques limit multiplexing to 4-5 targets, hindering comprehensive analysis.
Approach:
- Developed a robust hybridization chain reaction (HCR) spectral imaging method.
- Integrated linear unmixing to resolve overlapping fluorophore signals.
- Applied the technique to highly autofluorescent samples like whole-mount zebrafish embryos and mouse brain sections.
Key Points:
- Achieved simultaneous imaging of 10 RNA and/or protein targets.
- Demonstrated quantitative signal amplification and unmixing across 10 channels.
- Enabled subcellular resolution for relative quantitation and single-molecule resolution for absolute RNA quantitation.
Conclusions:
- HCR spectral imaging offers a unified framework for high-plex, quantitative imaging.
- Successfully overcomes limitations of autofluorescence and spectral overlap.
- Provides a powerful tool for high-resolution molecular and cellular analysis in complex biological tissues.
