AccScience Publishing / JBM / Online First / DOI: 10.14440/jbm.2025.0101
RESEARCH ARTICLE

3D visualization of human colon tissue using a modified CUBIC-based tissue-clearing technique

Pavel Pavlov1† Andreas Kontny1† Neele Wagner1 Nikola Kolev2 Alexander Zlatarov2 Turgay Kalinov2 Anton B. Tonchev1*
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1 Department of Anatomy and Cell Biology, Faculty of Medicine, Medical University Varna, Varna 9002, Bulgaria
2 Department of General and Operative Surgery, Faculty of Medicine, Medical University Varna, Varna 9002, Bulgaria
JBM 2025, 12(1), e99010052 https://doi.org/10.14440/jbm.2025.0101
Submitted: 15 October 2024 | Revised: 19 December 2024 | Accepted: 7 January 2025 | Published: 4 February 2025
© 2025 by the Author(s). This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
Abstract

Background: Colorectal cancer represents one of the most common neoplastic diseases worldwide, making it a frequent focus in routine pathological analyses. Visualizing complex three-dimensional (3D) structures, such as nerves within tumors, requires thick tissue sections, which necessitates the use of optical tissue-clearing methods to achieve transparency. However, following tissue clearing, samples typically require advanced imaging techniques such as light-sheet and two-photon confocal microscopy, which are usually unavailable in standard histological laboratories. Objective: We aimed to demonstrate how a well-established tissue-clearing approach can be adapted for use in a routine histological laboratory, enabling a robust 3D visualization of nerve fibers in samples of both normal human colon and colon cancer tissues. Methods: We modified the “clear unobstructed brain/body imaging cocktails” method, originally developed for whole-brain imaging in mice, and applied it to human colon tissue samples measuring approximately 10 mm3, a standard size typically processed in pathological laboratories. Results: Our protocol, which integrates a tissue-clearing technique, enabled reliable immunofluorescent visualization of colonic nerve fibers labeled with anti-β3-tubulin antibodies. The labeled nerve fibers could be observed using a standard epifluorescence microscope, and high-quality 3D reconstructions were generated through a simple image analysis approach using the open-source software ilastik, which eliminates the need for confocal microscopy. Conclusion: The proposed steps provide a valuable method for researchers to visualize complex 3D structures, such as neural cells and processes, in both normal and tumor-transformed tissue settings.

Keywords
Clear unobstructed brain/body imaging cocktails
CUBIC
Tissue clearing
3D-imaging
Colon
Colorectal cancer
Funding
This work was supported by the European Regional Development Fund through the Operational Program “Science and Education for Smart Growth” (contract #BG05M2OP001- 1.002-0010-C01), the European Commission Horizon 2020 Framework Program (Project 856871–TRANSTEM), and the MU-Varna intramural research grants (16007/2016 and 19012/2019).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be perceived as a potential conflict of interest.
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Journal of Biological Methods, Electronic ISSN: 2326-9901 Print ISSN: TBA, Published by POL Scientific