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HCR spectral imaging: 10-plex, quantitative, high-resolution RNA and protein imaging in highly autofluorescent
Samuel J Schulte1, Mark E Fornace1, John K Hall1
1Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Development (Cambridge, England)
|February 28, 2024
Summary
Hybridization chain reaction (HCR) spectral imaging enables simultaneous visualization of ten RNA and protein targets in challenging autofluorescent samples. This method provides quantitative, high-resolution imaging for biological research.
Area of Science:
- Molecular Biology
- Biotechnology
- Imaging Science
Background:
- Multiplex imaging of RNA and proteins is crucial for understanding biological processes.
- Conventional methods struggle with signal overlap and autofluorescence in complex samples.
- Spectral imaging offers potential for higher multiplexing but faces technical hurdles.
Purpose of the Study:
- To develop a robust method for high-plex, quantitative imaging in highly autofluorescent biological samples.
- To overcome limitations of conventional bandpass imaging and spectral imaging in complex tissues.
- To enable simultaneous detection of multiple RNA and protein targets with high resolution.
Main Methods:
- Utilized hybridization chain reaction (HCR) for signal amplification.
- Implemented spectral imaging combined with linear unmixing.
- Applied the technique to whole-mount zebrafish embryos and mouse brain sections.
Main Results:
- Achieved simultaneous imaging of ten RNA and/or protein targets.
- Demonstrated quantitative signal detection across all ten channels.
- Enabled subcellular resolution for relative quantitation and single-molecule resolution for absolute RNA quantitation.
- Successfully imaged targets in highly autofluorescent samples.
Conclusions:
- HCR spectral imaging with linear unmixing is a powerful tool for multiplex, quantitative imaging.
- This method significantly advances the ability to study gene and protein expression in complex biological systems.
- The technique provides high-resolution molecular insights in anatomically relevant contexts.
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