Light Sheet Analysis of In-vitro Wound Healing

Abstract

Collective cell migration and tissue expansion are integral to wound healing. Studies into the dynamics of these phenomena primarily utilize 2D microscopy techniques with 3D microscopy techniques often being too phototoxic for the long time-lapses necessary to gather sufficient resolution in the z-axis. With advances in light sheet fluorescence imaging in the form of dual-view inverted selective plane illumination microscopy (diSPIM), growing epithelial tissues can be rapidly imaged with high resolution and minimal photo-toxicity, offering new insights into the dynamics of cell sheets and morphology of such tissues. Upon Princeton University’s recent procurement of a diSPIM, its proper calibration and the establishment of run conditions became necessary and thus are the focus of this thesis. Using micropatterned epithelial (MDCK-II) monolayers, the diSPIM was optimized for capturing live 3D time-lapses, imaging colliding epithelial tissues in 3D, and exploring the effects of shape and orientation on the collision dynamics of tissue collision. From this work, it was found that by orienting colliding tissues at a 45º angle from one another, a smooth collision boundary that is distinct from rough head-on collision boundaries within the same tissue can be created. The smooth boundaries were found in various tissue shapes and collision conformations, suggesting that the observed phenomenon is dependent on the curvature of the collision rather than the overall shape of the tissue. Additionally, we were able to repeatably induce the formation of lines of tissue between two confining epithelial monolayers, structures of approximately a single cell in width, known as an "escape."