Non-invasive characterization of structure and morphology of silk fibroin biomaterials using non-linear microscopy

Designing biomaterial scaffolds remains a major challenge in tissue engineering. Key to this challenge is improved understanding of the relationships between the scaffold properties and its degradation kinetics, as well as the cell interactions and the promotion of new matrix deposition. Here we present the use of non-linear spectroscopic imaging as a non-invasive method to characterize not only morphological, but also structural aspects of silkworm silk fibroin-based biomaterials, relying entirely on endogenous optical contrast. We demonstrate that two photon excited fluorescence and second harmonic generation are sensitive to the hydration, overall beta sheet content and molecular orientation of the sample. Thus, the functional content and high resolution afforded by these non-invasive approaches offer promise for identifying important connections between biomaterial design and functional engineered tissue development. The strategies described also have broader implications for understanding and tracking the remodeling of degradable biomaterials under dynamic conditions both in vitro and in vivo.