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Recent efforts have integrated tissue-clearing techniques with fluorescent labeling through click chemistry, allowing high-resolution three-dimensional fluorescence imaging. Nevertheless, these techniques have faced a challenge in limited staining depth, confining their use to imaging tissue sections or partial organs. In this study, we introduce Click3D, a method for thoroughly staining whole organs using click chemistry. We identified click reaction conditions that improve staining depth with our custom-developed assay. The Click3D protocol exhibits a greater staining depth compared to conventional methods. Using Click3D, we have successfully achieved whole-kidney imaging of nascent RNA and whole-tumor imaging of hypoxia. We have also accomplished whole-brain imaging of hypoxia by using the clickable hypoxia probe, which has a small size and, therefore, has high permeability to cross the blood-brain barrier.<\/jats:p>","DOI":"10.1126\/sciadv.ado8471","type":"journal-article","created":{"date-parts":[[2024,7,17]],"date-time":"2024-07-17T18:00:47Z","timestamp":1721239247000},"update-policy":"https:\/\/doi.org\/10.34133\/aaas_crossmark","source":"Crossref","is-referenced-by-count":21,"title":["Click3D: Click reaction across deep tissues for whole-organ 3D fluorescence imaging"],"prefix":"10.1126","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4736-2922","authenticated-orcid":true,"given":"Iori","family":"Tamura","sequence":"first","affiliation":[{"name":"Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, 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