Optical microscopy encompasses high-resolution imaging techniques that can be used to non-destructively investigate and characterize living biological systems and engineered tissue constructs in culture. In particular, nonlinear optical microscopy (NLOM) is well suited for the visualization and quantification of processes involved in cell-extracellular matrix interactions in vivo. Current NLOM technology enables concomitant molecular imaging and visualization of microstructural organization that could provide a direct link between signal transduction and biological effect at microscopic length scales that culminate into tissue macroscopic properties and function. This review highlights the fundamentals of nonlinear optical interactions between light and tissue and presents a direction for future technology development to better complement quantitative, high-throughput assays of the modern life sciences.