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Published January 17, 2024 | in press
Journal Article Open

Multiplex, Quantitative, High-Resolution Imaging of Protein:Protein Complexes via Hybridization Chain Reaction

  • 1. ROR icon California Institute of Technology


Signal amplification based on the mechanism of hybridization chain reaction (HCR) facilitates spatial exploration of gene regulatory networks by enabling multiplex, quantitative, high-resolution imaging of RNA and protein targets. Here, we extend these capabilities to the imaging of protein:protein complexes, using proximity-dependent cooperative probes to conditionally generate a single amplified signal if and only if two target proteins are colocalized within the sample. HCR probes and amplifiers combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind nonspecifically in the sample, they will not generate amplified background. We demonstrate protein:protein imaging with a high signal-to-background ratio in human cells, mouse proT cells, and highly autofluorescent formalin-fixed paraffin-embedded (FFPE) human breast tissue sections. Further, we demonstrate multiplex imaging of three different protein:protein complexes simultaneously and validate that HCR enables accurate and precise relative quantitation of protein:protein complexes with subcellular resolution in an anatomical context. Moreover, we establish a unified framework for simultaneous multiplex, quantitative, high-resolution imaging of RNA, protein, and protein:protein targets, with one-step, isothermal, enzyme-free HCR signal amplification performed for all target classes simultaneously.

Copyright and License

© 2024 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0.


We thank M. E. Bronner for reading a draft of the manuscript and G. Shin of the Molecular Technologies resource within the Beckman Institute at Caltech for providing HCR reagents. This work was funded by the National Institutes of Health (NIBIB R01EB006192 and NIGMS training grant GM008042 to S.J.S.) and by the Beckman Institute at Caltech (Programmable Molecular Technology Center, PMTC). The Leica Stellaris 8 confocal microscope in the Biological Imaging Facility within the Beckman Institute at Caltech was purchased with support from Caltech and the following Caltech entities: the Beckman Institute, the Resnick Sustainability Institute, the Division of Biology and Biological Engineering, and the Merkin Institute for Translational Research.

Conflict of Interest

The authors declare the following competing financial interest(s): Patents, pending patent applications, and the startup company Molecular Instruments.



Additional details

January 31, 2024
January 31, 2024