Pavlov Pavel, Kontny Andreas, Wagner Neele, Kolev Nikola, Zlatarov Alexander, Kalinov Turgay, Tonchev Anton B
Department of Anatomy and Cell Biology, Faculty of Medicine, Medical University Varna, Varna 9002, Bulgaria.
Department of General and Operative Surgery, Faculty of Medicine, Medical University Varna, Varna 9002, Bulgaria.
J Biol Methods. 2025 Feb 4;12(1):e99010052. doi: 10.14440/jbm.2025.0101. eCollection 2025.
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.
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.
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 mm, a standard size typically processed in pathological laboratories.
Our protocol, which integrates a tissue-clearing technique, enabled reliable immunofluorescent visualization of colonic nerve fibers labeled with anti-β-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.
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.
结直肠癌是全球最常见的肿瘤性疾病之一，因此它经常是常规病理分析的重点。要可视化复杂的三维（3D）结构，如肿瘤内的神经，需要较厚的组织切片，这就需要使用光学组织透明化方法来实现透明。然而，在组织透明化之后，样本通常需要先进的成像技术，如光片显微镜和双光子共聚焦显微镜，而这些技术在标准组织学实验室中通常无法获得。
我们旨在展示一种成熟的组织透明化方法如何适用于常规组织学实验室，从而能够对正常人类结肠和结肠癌组织样本中的神经纤维进行可靠的3D可视化。
我们修改了最初为小鼠全脑成像开发的“清除无阻脑/体成像混合液”方法，并将其应用于尺寸约为10毫米的人类结肠组织样本，这是病理实验室通常处理的标准尺寸。
我们的方案整合了一种组织透明化技术，能够可靠地对用抗β-微管蛋白抗体标记的结肠神经纤维进行免疫荧光可视化。使用标准的落射荧光显微镜可以观察到标记的神经纤维，并通过使用开源软件ilastik的简单图像分析方法生成高质量的3D重建图像，从而无需使用共聚焦显微镜。
所提出的步骤为研究人员在正常和肿瘤转化组织环境中可视化复杂的3D结构（如神经细胞和突起）提供了一种有价值的方法。
