Mechanosensitive ion channels play an important role for the perception of mechanical signals such as touch, balance, or sound. Here, a new experimental strategy is presented providing well-defined access to single mechanosensitive ion channels in living cells. As a representative example, the investigation of mechanosensitive transduction channels in cochlear hair cells is discussed in detail including all essential technical aspects. Three different techniques were combined: atomic force microscopy (AFM) as a device for local mechanical stimulation, patch clamp for recording the current response of mechanosensitive ion channels, and differential interference contrast (DIC) microscopy equipped with an upright water-immersion objective lens. A major challenge was to adapt the mechanical design of the AFM setup to the small working distance of the light microscope and the electrical design of the AFM electronics. Various protocols for the preparation and investigation of the organ of Corti with AFM are presented.