Stem cell technologies hold great promise for engineering replacement tissues for repairing functional loss from trauma or disease. Such therapies are particularly important for replacing bone and cartilage in the aging population to maintain an active quality of life. However, the application of stem cells to generate individualized implantable grafts suffers from patient-to-patient variability that is unpredictable and immeasurable without destructive techniques, representing a major bottleneck in translating stem cell technologies to the clinic and delivering a quality product. During tissue formation, cells deposit extracellular matrix molecules that possess a unique fluorescence signature, which can be detected by light, while matrix quantity, detectable by ultrasound, correlates with mechanical strength. During the reporting period, our team has combined existing instrumentation with custom-made parts to realize a multimodal imaging platform using light and sound, capable of acquiring time-resolved fluorescence spectroscopy (TRFS), fluorescence lifetime imaging (FLIM) and ultrasound backscatter microscopy (UBM) measurements in a fast and repeatable manner. We completed three validation studies using well-defined biomaterials and native articular cartilage to develop correlations between optical signatures and mechanical properties. By inducing different kinds of collagen crosslinks with macromolecules, we demonstrated that changes in fluorescence lifetime (i.e., the duration of excitement by light) could be detected by FLIM, and these differences were reflected in increased mechanical properties of the hydrogels. Fluorescence imaging and UBM could also detect changes in articular cartilage enzymatically treated to remove collagen and collagen crosslinks, and these data correlated well with reductions in mechanical properties and loss of tissue observed by histology. Collectively, the progress on this project demonstrates the ability of optical imaging to discern additive or reductive characteristics in natural materials and native cartilage.