Cell substrate interactions play an important role in regulating cellular physiological and pathological processes, and therefore, investigating cell substrate interface is meaningful for understanding the behaviors of cells. However, so far, the underlying mechanisms which guide the nanoscopic biological activities taking place at the cell-substrate interface remain poorly understood. The advent of atomic force microscopy (AFM) provides a powerful tool for characterizing the structures and properties of native biological and biomaterial systems with unprecedented spatiotemporal resolution, which offers new possibilities for understanding the physical sciences of biomaterials. Here, AFM was utilized to unravel the nanotopographical surfaces for regulating cellular behaviors on three different substrates (glass slide, mica, and Petri dish). First, the decellularized substrates prepared with the use of ammonia and trypsin were imaged by AFM, significantly showing the nanogranular substances on the decellularized substrates as well as the cell membrane patches for uncovering the detailed situations of mechanical contact between cells and substrates. Next, experiments performed on chemically fixed substrates with the use of paraformaldehyde together with AFM time-lapse imaging remarkably showed that nanogranular depositions from the cell culture medium appeared on the substrates for promoting cell growth. Further, the detailed cell culture medium components which contribute to the nanogranular depositions are identified. Finally, the dynamic alterations in surface roughness and mechanical properties of substrates and cells during cell growth were quantitatively measured by AFM, revealing the diverse changes of the multiple physical properties (surface roughness, adhesion force, Young's modulus, and relaxation time) during cell-substrate interactions. The research provides novel insights into the nanotopographical surfaces for cell-substrate interactions, which will be useful for understanding cellular behaviors.