Volumetric Cyclic Immunofluorescence for 3D Spatial Profiling of Tumour, Stroma, and Immune Structures in Human FFPE Tissue
- 1Harvard Medical School
Abstract
Mapping how parenchymal, immune, and stromal cells interact with extended structures such as blood vessels and nerves is challenging using conventional thin-section tissue imaging. Volumetric methods such as light-sheet fluorescence microscopy (LSFM) are ideal in these cases and LSFM is widely used in animal models such as fish and mice. This is enabled by well-established clearing methods, genetically encoded reporters, and vascular dyes. However, LSFM is rarely utilized for human tissue, which is primarily available as formaldehyde-fixed paraffin embedded (FFPE) specimens. Here, we present an approach to volumetric cyclic immunofluorescence (v-CyCIF) that enables 40-plex or greater imaging of immune and other cell types in human specimens up to 1 mm thick. We demonstrate the use of v-CyCIF to study neuroimmune interactions and lymphoid structures in normal and cancer tissues. Following LSFM imaging, specimens can be re-embedded and sectioned to ~50 {micro}m thick for high-resolution confocal imaging of punctate structures and cell-cell junctions. v-CyCIF therefore provides a flexible framework for multi-scale 3D profiling of clinical specimens across imaging formats and resolutions.
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BibTeX
@misc{wong2026volumetric,
title = {{Volumetric Cyclic Immunofluorescence for 3D Spatial Profiling of Tumour, Stroma, and Immune Structures in Human FFPE Tissue}},
author = {A. Y. H. Wong and Y. D. Lu and Z. Zhao and S. W. Choi and H. Park and F. Zhou and Z. Maliga and Y. Anang and S. Rastgou Talemi and S. Coy and G. Danuser and S. Santagata and C. Yapp and P. K. Sorger},
howpublished = {bioRxiv},
year = {2026},
month = may,
doi = {10.64898/2026.05.17.725158},
url = {https://www.biorxiv.org/content/10.64898/2026.05.17.725158v3},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Volumetric Cyclic Immunofluorescence for 3D Spatial Profiling of Tumour, Stroma, and Immune Structures in Human FFPE Tissue
AU - A. Y. H. Wong
AU - Y. D. Lu
AU - Z. Zhao
AU - S. W. Choi
AU - H. Park
AU - F. Zhou
AU - Z. Maliga
AU - Y. Anang
AU - S. Rastgou Talemi
AU - S. Coy
AU - G. Danuser
AU - S. Santagata
AU - C. Yapp
AU - P. K. Sorger
PY - 2026
DA - 2026/05/17
PB - bioRxiv
DO - 10.64898/2026.05.17.725158
UR - https://www.biorxiv.org/content/10.64898/2026.05.17.725158v3
AB - Mapping how parenchymal, immune, and stromal cells interact with extended structures such as blood vessels and nerves is challenging using conventional thin-section tissue imaging. Volumetric methods such as light-sheet fluorescence microscopy (LSFM) are ideal in these cases and LSFM is widely used in animal models such as fish and mice. This is enabled by well-established clearing methods, genetically encoded reporters, and vascular dyes. However, LSFM is rarely utilized for human tissue, which is primarily available as formaldehyde-fixed paraffin embedded (FFPE) specimens. Here, we present an approach to volumetric cyclic immunofluorescence (v-CyCIF) that enables 40-plex or greater imaging of immune and other cell types in human specimens up to 1 mm thick. We demonstrate the use of v-CyCIF to study neuroimmune interactions and lymphoid structures in normal and cancer tissues. Following LSFM imaging, specimens can be re-embedded and sectioned to ~50 {micro}m thick for high-resolution confocal imaging of punctate structures and cell-cell junctions. v-CyCIF therefore provides a flexible framework for multi-scale 3D profiling of clinical specimens across imaging formats and resolutions.
ER -