Integrin-mediated cell-matrix adhesion complexes (CMACs) are important modulators of cell adhesion and migration and are key transducers of cell signaling events. In cell migration, correct coordination of CMAC adhesive strength and signaling is pivotal. Cell migration has been extensively studied in 2D cell culture systems, and while studies in more physiological 3D environments are still rudimentary, it is already clear that CMACs behave very differently in 3D environments as compared to 2D. This field is particularly lacking in high resolution characterizations of CMAC dynamics during 3D-migration. By utilizing a cutting edge 3D mirofluidic chamber amenable to high resolution imaging, matched with the latest imaging and 3D matrix technologies, I aim to address this significant gap in knowledge. I will characterize CMAC number, distribution, size, shape, composition and molecular turnover in 3D and over time in the context of various 3D matrix environments and cell guidance cues. I will also study the effects of known mediators of CMAC function including c-Src, Focal adhesion kinase (FAK) and p21 activated kinase 4 (PAK4); their potential roles in CMAC dynamics and migration within 3D environments is unknown. Lastly, these imaging based experiments will be transferred into an ex vivo tissue environment to ascertain physiological significance. Overall, this project will provide significant insight into CMAC function during cell migration through a 3D substrate and make available new experimental tools and protocols for high resolution imaging in 3D matrices.