Novel imaging approach for simultaneous tracking of cell dynamics in distinct tissue layers reveals cells involved in colonic peristalsis

We have developed a novel approach for high-resolution confocal imaging across multiple tissue planes simultaneously. By combining confocal microscopy, piezo actuators, and optogenetic sensors, we can simultaneously capture images of dynamic fluorescence signals from various cell populations in different tissue layers (Z planes). This enables the decoding of cell-to-cell communication through complex tissues, offering a significant advancement in understanding how cells in distinct layers of tissue communicate and coordinate their functions and produce integrated behaviors. For example, our technique sheds light on myogenic coordination underlying colonic motility. Examining various cell types, such as interstitial cells of Cajal (ICC) and smooth muscle cells (SMC), distributed through the thickness of muscle layers, we demonstrate distinct Ca²⁺ signaling patterns and organization that underlie complex colonic motor activities.