Abstract Corneal scarring is a leading cause of blindness worldwide. Through their interactions with the extracellular matrix (ECM), stromal keratocytes play a central role in fundamental biological processes such as developmental morphogenesis and wound healing. Recent studies suggest that feedback from the ECM can have a significant impact stromal cell behavior. Specifically, ECM stiffness, topography and protein composition have all been shown to modulate keratocyte differentiation, contractility and patterning. However, there are still significant gaps in our knowledge of how multiple stimuli are integrated to produce specific cell phenotypes. The parent R01 project focuses on the development and testing of novel 2D and 3D experimental platforms which allow us to systematically incorporate combinations of both soluble and non-soluble cues. In particular, in Aim 1 we are determining how substrate elasticity modulates the keratocyte response to soluble biochemical cues; in Aim 2 we are determining how substrate topography and composition modulates the keratocyte response to biochemical and biomechanical cues; and in Aim 3 we are determining how substrate dimensionality (2-D versus 3-D) modulates the keratocyte response to biochemical, biomechanical and topographic cues. In this proposal we propose the acquisition of a Zeiss microscope stage CO2 incubator system that will be installed on a motorized Zeiss Axio Observer Z1 inverted microscope. This upgrade will allow us to perform live- cell analysis, time lapse imaging, and provide us with multi-modal information. Based on experimental data collected over the first 4 years of the project, our needs for live-cell and time lapse imaging have expanded dramatically. This instrumentation will allow us quantitatively (i) determine and measure dynamic cell morphology and transient cell activation information with improved temporal resolution; (ii) characterize the impact of collagen fibril alignment and topography on keratocyte migration in response to novel wound injury models we have developed; (iii) allow us to identify the relative roles of proliferation and migration in response to secreted and/or immobilized growth factors and extracellular matrix proteins; and (iv) allow us to determine the effects of 3D confinement on keratocyte migration. The acquisition of this live-cell imaging system is expected to provide unprecedented insights into the role of key biophysical signaling pathways on the differentiation of keratocytes (e.g. quiescent, migratory, regenerative and repair phenotypes). Knowledge obtained using these novel constructs should also aid in the development of targeted anti-fibrosis therapies, as well as guide tissue engineering approaches to developing stromal tissue replacements.