Click3D: Click reaction across deep tissues for whole-organ 3D fluorescence imaging.

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.

Pimonidazole-alkyne conjugate for sensitive detection of hypoxia by Cu-catalyzed click reaction.

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye.

Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing.

Elucidation of biological phenomena requires imaging of microenvironments in vivo. Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.