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.

中文翻译：

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Click3D：深层组织的点击反应，用于全器官 3D 荧光成像


点击化学通过有效的生物正交反应提供各种应用。在生物成像中，经常使用预靶向策略，利用带有点击手柄的分子探针和使它们可观察的报告分子之间的点击反应。最近的努力通过点击化学将组织透明化技术与荧光标记相结合，从而实现高分辨率三维荧光成像。然而，这些技术面临着染色深度有限的挑战，仅限于对组织切片或部分器官进行成像。在这项研究中，我们介绍了 Click3D，一种使用点击化学对整个器官进行彻底染色的方法。我们通过定制开发的检测确定了可提高染色深度的点击反应条件。与传统方法相比，Click3D 方案表现出更大的染色深度。利用Click3D，我们成功实现了新生RNA的全肾成像和缺氧的全肿瘤成像。我们还通过使用可点击的缺氧探针完成了缺氧的全脑成像，该探针尺寸小，因此具有较高的穿过血脑屏障的渗透性。