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Atomic force acoustic microscopy reveals the influence of substrate stiffness and topography on cell behaviorThe stiffness and the topography of the substrate at the cell-substrate interface are two key properties influencing cell behavior. In this paper, atomic force acoustic microscopy (AFAM) is used to investigate the influence of substrate stiffness and substrate topography on the responses of L929 fibroblasts. This combined nondestructive technique is able to characterize materials at high lateral resolution. To produce substrates of tunable stiffness and topography, we imprint nanostripe patterns on undeveloped and developed SU-8 photoresist films using electron-beam lithography (EBL). Elastic deformations of the substrate surfaces and the cells are revealed by AFAM. Our results show that AFAM is capable of imaging surface elastic deformations. By immunofluorescence experiments, we find that the L929 cells significantly elongate on the patterned stiffness substrate, whereas the elasticity of the pattern has only little effect on the spreading of the L929 cells. The influence of the topography pattern on the cell alignment and morphology is even more pronounced leading to an arrangement of the cells along the nanostripe pattern. Our method is useful for the quantitative characterization of cell-substrate interactions and provides guidance for the tissue regeneration therapy in biomedicine.