In Vivo Click Chemistry Enables Multiplexed Intravital Microscopy

Abstract

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.

Intravital microscopy for real-time monitoring of drug delivery and nanobiological processes

“…In the current perspective, this asset will gain further potential when accompanied by functional reporters. Especially the advent of multiplex labelling of window chambered tumor models [160], which is akin to in vivo flow cytometry, may open up further opportunities, especially for the study of the TME and other complex cellular milieus. This method utilizes a scission-accelerated fluorophore exchange (SAFE) to entirely remove a fluorophore from previously labeled cells within dorsal and cranial window chambers in mice.…”

Highly Active Myeloid Therapy for Cancer

“…Dorsal windows were implanted into IL-12 eYFP reporter mice. , All confocal images were collected by using a customized Olympus FV1000 confocal microscope (Olympus America). A 2× (XLFluor, N.A.…”

Cycling, Fasting, Fishing, and Other Things: The Emerging World of Biocompatible Chemistries

“…In a demonstration of how gentle bioorthogonal chemistries are for cellular multiplexing, researchers from the Weissleder lab performed cyclic imaging on living tissues directly in an implanted mouse glioblastoma using a variation of the SAFE method for intravital microscopy (SAFE‐IVM). [ 8 ] While this is not a practical technique for diagnosing and monitoring therapeutic response to cancer, in vivo imaging allowed the researchers to visualize tumor and immune responses in real time to gain a better understanding of what processes are occurring during the delivery of chemotherapy. The nontoxic nature of bioorthogonal chemistries provide a robust method for multiplexed tumor monitoring that would otherwise be impossible with comparative current techniques.…”