Click3D: Click reaction across deep tissues for whole-organ 3D fluorescence imaging
Iori Tamura, Daichi M. Sakamoto, 博志 末益, Yutaro Saito, Naoki Yamada, Jumpei Morimoto, Yoichi Takakusagi, Masafumi Kuroda, Shimpei I. Kubota, Hiroyuki Yatabe, Minoru Kobayashi, Hiroshi Harada, Kazuki Tainaka, Shinsuke Sando
Abstract
Click chemistry offers various applications through efficient bioorthogonal reactions. In bioimaging, pretargeting strategies have often been used, using click reactions between molecular probes with a click handle and reporter molecules that make them observable. 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.