ABSTRACT Hydrogels play a key role in tissue engineering, providing a supporting structure to mimic a native extracellular matrix (ECM) micro-environment for cell adhesion, proliferation and migration. As polymer networks infiltrated with water, hydrogels are similar with human body, constituting most of their cells, extracellular matrices, tissues and organs due to hydrophilic polymer networks infiltrated with water so that they have been widely applied for diabetic wound healing, bioadhesive sealant and wearable device interface in biomedical applications. An ideal engineered biomedical hydrogel's elastic properties should match well with the native tissue that it is being integrated. In biomedical applications, mechanical interactions with dissimilar mechanical stiffness between target tissues/organs and hydrogels can cause impaired functional efficacy such as foreign- body response, tissue damage or scar formation. In tissue engineering, the mechanical stiffness of hydrogels is dynamic because numerous features can influence their elastic properties such as cell adhesion, proliferation, migration, levels of polymer molecular weights, levels of cross-linked collagens and cell density. Therefore, characterizing hydrogel mechanical stiffness over time is critical. Mechanical tests such as dynamic mechanical analysis (DMA) are the standard method to characterize hydrogel mechanical stiffness. However, the major limitations of mechanical tests are that they are destructive measurements, multiple replicate samples are required for studies with multiple time points and only global elasticity measurements are provided. These limitations lead to difficulties for exploring the optimization of mechanical stiffness of hydrogels to host tissues in biomedical and tissue engineering applications. In this proposal, 1) we will develop an ultrasound-based elasticity measurement technique named two-dimensional acoustic force elastography microscope (2D-AFEM), which can address current difficulties of mechanical tests and can repeatedly measure 2D (x, y) Young's modulus of thin-layer cell encapsulated hydrogels. 2) To study 2D-AFEM performance, we will test the 2D-AFEM elastography resolution, 2D Young's modulus of homogeneous and heterogeneous hydrogel scaffolds, and validate experimental results by numerical simulation and DMA. 3) To study how living cells affect stiffness changes of hydrogel scaffolds to obtain a desired functionality, we will use 2D-AFEM to explore elastic changes of cell encapsulated scaffolds and explore the relationship between elastic changes and biochemical markers over time for longitudinal studies. The proposed 2D-AFEM will be a promising modality to repeatedly and quickly evaluate 2D Young's modulus of living hydrogels over time to explore the best match of the elasticity to host tissues for various applications in biomedical, tissue engineering and biomaterial fields.