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From chemRxiv
University of Tokyo

Click3D: A whole-organ 3D imaging method utilizing tissue clearing-compatible click reaction

Click chemistry stands as an invaluable chemical technology, offering a multitude of applications through highly selective and efficient bioorthogonal reactions under biological environments. In the realm of bioimaging, pre-targeting strategies have often been employed, utilizing click reactions between molecular probes with a click handle and reporter molecules that make them observable. Recent efforts have integrated state-of-the-art tissue-clearing techniques with fluorescent labeling through click chemistry, allowing high-resolution 3D fluorescence imaging of the target molecules. Nevertheless, these techniques have faced a challenge in terms of limited staining depth, confining their use to imaging tissue sections or partial organs. In this study, we introduce Click3D, a method for thorough staining of whole tissues and whole organs using click chemistry. We identified click reaction conditions that improve staining depth with our custom-developed click staining depth assay. The Click3D protocol, optimized by incorporating these conditions, exhibits a substantially greater staining depth compared to conventional methods. Through the implementation of Click3D, we have successfully achieved whole-kidney imaging of nascent RNA and whole-tumor imaging of hypoxia. Remarkably, we have also accomplished whole-brain imaging of hypoxia by employing the clickable hypoxia probe, which has a small size and, therefore, has high permeability to cross the blood-brain barrier. The Click3D method, leveraging the versatility of click chemistry, is a foundational technology and is expected to have diverse applications employing various clickable probes.
Published on February 12, 2024
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