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

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.