Abstract
Intravital microscopic imaging (IVM) allows for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo. Current IVM strategies primarily use fluorescence imaging; however, with the advances in bioluminescence imaging and the development of new bioluminescent reporters with expanded emission spectra, the applications for bioluminescence are extending to single cell imaging. Herein, we describe a molecular imaging window chamber platform that uniquely combines both bioluminescent and fluorescent genetically encoded reporters, as well as exogenous reporters, providing a powerful multi-plex strategy to study molecular and cellular processes in real-time in intact living systems at single cell resolution all in one system. We demonstrate that our molecular imaging window chamber platform is capable of imaging signaling dynamics in real-time at cellular resolution during tumor progression. Importantly, we expand the utility of IVM by modifying an off-the-shelf commercial system with the addition of bioluminescence imaging achieved by the addition of a CCD camera and demonstrate high quality imaging within the reaches of any biology laboratory.
Highlights
Advances that allow for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo have become crucial
Advances in single cell digital techniques have laid the foundational framework for advances in cancer immunology that account for cellular heterogeneity; these techniques are destructive and time-course dynamics require a large number of subjects for analysis of genetic, molecular and cellular changes that cannot be tracked or sampled temporally or spatially within the same individual
A variety of custom-built intravital microscopy systems have been reported by expert laboratories [1,2,3,4,5,6,7,40,41,42,53,54,55,56,57,58,59,60,61,62]
Summary
Intravital microscopic imaging (IVM) has proven to be a powerful imaging technique [1,2,3,4,5,6,7,8,9,10,11], one of the few molecular imaging strategies that provides both spatial and temporal information regarding heterogeneous living systems in real-time at cellular resolution This technology has become increasingly critical in studies of the tumor immune microenvironment, as it has become increasingly recognized that the immune system plays complex dual roles in cancer, both beneficially and adversely impacting tumorigenesis [12,13,14,15]. Enormous advances in immunology have been achieved though the analysis of tissues in bulk at different stages of tumor progression as well as a variety of in vitro and ex vivo methodologies Many of these findings provide only a snapshot in time of orchestrated molecular and cellular changes occurring over time. Few imaging modalities can achieve noninvasive cellular resolution sufficient to image the molecular mechanisms of cell trafficking, cell–cell interactions and the associated molecular signals that IVM provides
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