In Vivo Click Chemistry Enables Multiplexed Intravital Microscopy

• Published in: Advanced science 2022-08, Vol.9 (24), p.e2200064-n/a
• Main Authors: Ko, Jina, Lucas, Kilean, Kohler, Rainer, Halabi, Elias A., Wilkovitsch, Martin, Carlson, Jonathan C. T., Weissleder, Ralph
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; cancer; click chemistry; Click Chemistry - methods; drug delivery; Experiments; Fluorescent Dyes - chemistry; Intravital Microscopy; Kinetics; Longitudinal studies; Mice; Microscopy; Proteins; Reagents; Staining and Labeling; Stains & staining
• ISSN: 2198-3844; 2198-3844
• DOI: 10.1002/advs.202200064

The ability to observe cells in live organisms is essential for understanding their function in complex in vivo milieus. A major challenge today has been the limited ability to perform higher multiplexing beyond four to six colors to define cell subtypes in vivo. Here, a click chemistry‐based strategy is presented for higher multiplexed in vivo imaging in mouse models. The method uses a scission‐accelerated fluorophore exchange (SAFE), which exploits a highly efficient bioorthogonal mechanism to completely remove fluorescent signal from antibody‐labeled cells in vivo. It is shown that the SAFE‐intravital microscopy imaging method allows 1) in vivo staining of specific cell types in dorsal and cranial window chambers of mice, 2) complete un‐staining in minutes, 3) in vivo click chemistries at lower (µm) and thus non‐toxic concentrations, and 4) the ability to perform in vivo cyclic imaging. The potential utility of the method is demonstrated by 12 color imaging of immune cells in live mice. A click chemistry‐based technology that enables multiplexed in vivo imaging in mouse models is presented. This method uses a scission‐accelerated fluorophore exchange, which completely removes fluorescent signal from antibody‐labeled cells in vivo, allowing cyclic imaging for high multiplexing.