ABSTRACT Multiplexed analytical techniques such as flow cytometry, scRNAseq and spatial transcriptomics have revolutionized modern biology. Unfortunately, there are still no reliable ways of performing highly multiplexed in vivo real-time imaging to understand the functional behavior of cells and molecules in their intact microenvironment over time. Based on advances in “click chemistries” we have developed a novel, scission based in vivo multiplexing technology (SAFE; Ko et al., 2022, Nat Biotech, in press; Ko et al., Adv Science, 2022 in press), that for the first time allows intravital imaging far beyond what is currently possible (~12-20 channel in vivo imaging in feasibility studies instead of 4-6 channels afforded by intravital microscopy). The new technology utilizes specially labeled fluorescent affinity ligands i) where an exogenously administered tetrazine cleaves a C2TCO containing linker between antibody and fluorochromes in seconds and at non-toxic concentrations and ii) allows antibody-tagged superbright polymeric and indocyanine dyes to be washed away between cycles of imaging and thus re-staining. The goal of this proposal is to further develop and validate the technology and then use it to gain deeper temporal and spatial insight into the tumor microenvironment. We propose two specific aims: i) Optimize the SAFE-IVM technology for broader (~30 channel) multiplexed in vivo imaging (new chemistry including novel iTCO, Tz-BHQ, expand colors, improve imaging chambers); and ii) perform multiplexed imaging of tumor microenvironment and its immune cells. Findings from latter studies will be important because they will i) establish the utility of the new technology benchmarked against gold standards and ii) open the door to broader applicability of the new technology.