| Summary: | A major obstacle in studying the interplay between cancer cells and the immune system has been the examination of proposed biological pathways and cell interactions in a dynamic, physiologically relevant system in vivo. Intravital imaging strategies are one of the few molecular imaging techniques that can follow biological processes at cellular resolution over long periods of time in the same individual. Bioluminescence imaging has become a standard preclinical in vivo optical imaging technique with ever-expanding versatility as a result of the development of new emission bioluminescent reporters, advances in genomic techniques, and technical improvements in bioluminescence imaging and processing methods. Herein, we describe an advance of technology with a molecular imaging window chamber platform that combines bioluminescent and fluorescent reporters with intravital macro-imaging techniques and bioluminescence spectral unmixing in real time applied to heterogeneous living systems in vivo for evaluating tumor signaling dynamics and immune cell enzyme activities concurrently.
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