Examination of validity of sample preparation method for speed of sound evaluation in ultra-high frequency band

Scanning acoustic microscopy (SAM) system allows acoustic characterization at the cell size level . However, since ultrasound waves of several hundred MHz are used, the attenuation due to the propagation of ultrasound waves is large, and it is necessary to slice the measurement sample in about 10 μm. There are two standard methods of preparing thin sliced samples, paraffin-embedded and frozen, and we have confirmed that the time of formalin fixation, which is often used in both methods, affects the speed of sound (SoS). In addition, the paraffin-embedded thin sliced samples have the problem of melting fat in the tissues. On the other hand, the freezing method is the absence of heat treatment, so it might be possible to evaluate the SoS of fat. This paper investigated the effect of the sample preparation on evaluating SoS in the freezing method. In particular, we focused on the half Karnovsky fixation method and examined the effect of the different fixation methods on the SoS. Half Karnovsky fixative contains 0.5 % glutaraldehyde, 2 % paraformaldehyde, 30 mM HEPES buffer and is commonly used in an electron microscope.