High-plex protein imaging across thick tissue by synchronized amplification
- 1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, 02215, USA; Department of Systems Biology, Harvard Medical School, Boston,
Abstract
High-plex protein imaging is essential for decoding complex tissue organization, yet it remains largely confined to thin sections. Current approaches in millimeter-thick samples suffer from poor signal penetration and depth-dependent, uneven signal amplification. Even with specialized instrumentation to facilitate probe diffusion, iterative millimeter-thick tissue workflows can take more than four days per staining round, and reported eleven-marker experiments extend beyond a month. We introduce Amplification by Cyclic Extension (ACE), a DNA-barcoded, synchronized in situ amplification strategy that uses thermally controlled cyclic extension to coordinate DNA concatemer growth across tissue depths. By synchronously activating and coordinating reaction progression throughout the sample, ACE enables controlled, uniform signal amplification across tissue depths. Using ACE, we demonstrate 63-plex protein and RNA imaging in thin sections, 22-plex protein imaging in 200 µm mouse brain sections, and 16-plex protein imaging in 1 mm-thick brain tissue encompassing over three million cells. For millimeter-thick tissues, ACE reduces per-round target-switching time from multiple days to less than eight hours, shifting volumetric high-plex protein imaging from months to days. Together, these results establish ACE as a synchronized amplification strategy that enables scalable high-plex protein imaging in thick tissues.
Related
- Magnetization Transfer Imaging Shows Tissue Abnormalities in the Reversible Penumbra
- ELiAH2: update of the atlas of E3 ligases in human tissues by integrating tissue-specificity and docking properties of ligands for targeted protein degradation
- Imaging biological tissue at extended depth using swept-source visible-light OCT
- Spatial transcriptomics profiling across tissue boundaries in the developing mouse brain
- Non-contact optical imaging of tissue deformation enables in vivo cardiovascular monitoring
BibTeX
@misc{sheng2026highplex,
title = {{High-plex protein imaging across thick tissue by synchronized amplification}},
author = {K. Sheng and H. Su and G. Gowri and M. Furman and M. Serrata and L. Du and C. George and M. Nofal and R. McMillan and K. Ma and X. Lun and H. Sasaki and J. Jeong and G. Pihan and P. Yin},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.12.757405},
url = {https://www.biorxiv.org/content/10.64898/2026.10.12.757405v1},
note = {Public domain},
}
RIS
TY - UNPB
TI - High-plex protein imaging across thick tissue by synchronized amplification
AU - K. Sheng
AU - H. Su
AU - G. Gowri
AU - M. Furman
AU - M. Serrata
AU - L. Du
AU - C. George
AU - M. Nofal
AU - R. McMillan
AU - K. Ma
AU - X. Lun
AU - H. Sasaki
AU - J. Jeong
AU - G. Pihan
AU - P. Yin
PY - 2026
DA - 2026/10/12
PB - bioRxiv
DO - 10.64898/2026.10.12.757405
UR - https://www.biorxiv.org/content/10.64898/2026.10.12.757405v1
AB - High-plex protein imaging is essential for decoding complex tissue organization, yet it remains largely confined to thin sections. Current approaches in millimeter-thick samples suffer from poor signal penetration and depth-dependent, uneven signal amplification. Even with specialized instrumentation to facilitate probe diffusion, iterative millimeter-thick tissue workflows can take more than four days per staining round, and reported eleven-marker experiments extend beyond a month. We introduce Amplification by Cyclic Extension (ACE), a DNA-barcoded, synchronized in situ amplification strategy that uses thermally controlled cyclic extension to coordinate DNA concatemer growth across tissue depths. By synchronously activating and coordinating reaction progression throughout the sample, ACE enables controlled, uniform signal amplification across tissue depths. Using ACE, we demonstrate 63-plex protein and RNA imaging in thin sections, 22-plex protein imaging in 200 µm mouse brain sections, and 16-plex protein imaging in 1 mm-thick brain tissue encompassing over three million cells. For millimeter-thick tissues, ACE reduces per-round target-switching time from multiple days to less than eight hours, shifting volumetric high-plex protein imaging from months to days. Together, these results establish ACE as a synchronized amplification strategy that enables scalable high-plex protein imaging in thick tissues.
SN - 2692-8205
ER -