Physical forces are transduced into chemical reactions, thereby ultimately making a large impact on the whole-animal level phenotypes such as homeostasis, development and behavior. To understand mechano-chemical transduction, mechanical input should be quantitatively delivered with controllable vibration properties-frequency, amplitude and duration, and its chemical output should be noninvasively quantified in an unconstrained animal. However, such an experimental system has not been established so far. Here, we develop a noninvasive and unconstrained mechanochemical imaging microscopy. This microscopy enables us to evoke nano-scale nonlocalized vibrations with controllable vibration properties using a piezoelectric acoustic transducer system and quantify calcium response of a freely moving C. elegans at a single cell resolution. Using this microscopy, we clearly detected the calcium response of a single interneuron during C. elegans escape response to nano-scale vibration. Thus, this microscopy will facilitate understanding of in vivo mechanochemical physiology in the future.