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Scanning acoustic microscopy is potentially a powerful tool for characterizing the elastic properties of soft biological tissues and cells. In this paper, we present a method, multi-layer phase analysis (MLPA), which can be used to extract local speed of sound values, for both thin tissue sections mounted on glass slides and cultured cells grown on cell culture plastic, with a resolution close to 1 μm. The method exploits the phase information that is preserved in the interference between the acoustic wave reflected from the substrate surface and internal reflections from the acoustic lens. In practice, a stack of acoustic images are captured beginning with the acoustic focal point 4 μm above the substrate surface and moving down in 0.1-μm increments. Scanning parameters, such as acoustic wave frequency and gate position, were adjusted to obtain optimal phase and lateral resolution. The data were processed offline to extract the phase information with the contribution of any inclination in the substrate removed before the calculation of sound speed. Here, we apply this approach to both skin sections and fibroblast cells, and compare our data with the V(f) (voltage versus frequency) method that has previously been used for characterization of soft tissues and cells. Compared with the V(f) method, the MPLA method not only reduces signal noise but can be implemented without making a priori assumptions with regards to tissue or cell parameters.