Multiple reflections in three-dimensional images of scanning acoustic microscopy indicate fine cell-cell spatial interrelationship

Scanning acoustic microscopy is an effective tool for non-invasive observation of living cells’ elasticity based on acoustic impedance (AI). This tool allows the visualization of intracellular three-dimensional structures in the depth direction. Here, we investigated the mechanism of the appearance of artifactual AI images in three-dimensional tomography using various cellular conditions. These ghost AI images were derived from prolonged reflection waves in the time domain, and multiple reflections between cell-cell gap structure and intracellular elastic materials were synthesized, causing complex ghost images. Synthesized reflection waves contained information on specific intracellular spaces and the distribution of intracellular elastic components. The ghost images derived from reflection waves were weak in non-biological interactions. Our result suggests complex reflections of AI would not only be a ghost-image maker but also transfer information about invisible delicate structures in cultured cells. This reflection analysis would be useful for detecting artificial organs’ differentiation and cell-cell connection.