# An Open Access Chamber Designed for the Acoustic Characterisation of Microbubbles
## 3 Results  
For every acoustic scattering measurement presented in this section, a two-tailed t-test was performed between the average RMS amplitude component of the received data corresponding to a scattering measurement and a reference measurement in which no microbubbles were present. The null hypothesis that \"the introduction of microbubbles into the acoustic path has no effect on the amplitude component at frequency \(f_{s}\)\" was rejected at the 5% significance level for all of the amplitude components used to determine the scattering characteristics in the remainder of this section. This gives confidence that the MACC is suitable for performing microbubble scattering measurements. Figure 6 shows the fundamental backscatter calculated over the frequency range of 1.6-6.4 MHz using Equation (1). The mean backscatter values from Figure 6a are included in Appendix Table 1 and for Figure 6b in Appendix Table 12. From Figure 6, it can be seen that the scattering from SonoVue(r) is highest at the lowest frequencies investigated. This is in general agreement with previous studies [31]. As is expected, because the measured scattering is the summation of the scattering from individual microbubbles, the lower dilution sample resulted in a lower scattering value. Both dilutions show the same general trend with frequency, and this gives confidence that the chamber is suitable for the measurement of dilutions of SonoVue(r) in the range of clinical doses. The measurements from the various transducers also overlap and are in general agreement with regards to the overall trend. For the pairs of transducers that are used to investigate the same frequency range, the scattering measurements are in agreement. This is further validation that the measurements are reliable. There is some discrepancy between the scattering values at the frequencies that overlap between different transducer pairs. This is likely due to mismatches in the transducer sensitivity compounded by the limits of the usable bandwidth for a transducer. This highlights the importance of performing the experiment within reasonable frequency limits for a given transducer and in the robust calibration of transducer receive-sensitivity. Figure 6: Measured backscatter at the fundamental frequency over the frequency range of 1.6–6.4 MHz for (**a**) the 1:1000 and (**b**) the 1:2500 dilutions of SonoVue®. Measurement scattering signals were recorded using the transmit transducer as a receiver. The error bars reflect the standard error over the 12 experiment repetitions. Figure 7 shows the average RMS pressure spectrum obtained using various transducers from the first acquisition and for a transmit frequency of 1.6 MHz transmitted using a 2.25 MHz transducer (T1 in Figure 4). The spectrum measured using the inline 2.25 MHz transducer (T2 in Figure 4) for the reference measurement is included in Figure 7a. Figure 7b shows the backscattered data received by the transmit transducer (T1 in Figure 4) at the fundamental frequency and second harmonic. The second harmonic response is very low. This can be accounted for due to the limited bandwidth of the transducer. Figure 7c shows the data received by a 3.5 MHz transducer (T3 in Figure 4). In this case, the fundamental response is low, but the second harmonic is more pronounced. Figure 7d shows the combination of the fundamental response recorded by the transmit transducer and the second harmonic recorded by the 3.5 MHz transducer. It highlights how the signal received on multiple transducers can be combined to obtain the frequency response over a broader range of frequencies than would be possible with just a single receive transducer. It should be noted that the fusion of data from different receivers relies on the ability to obtain an accurate RMS pressure value for the receiver amplitude. Figure 8 shows the second harmonic scattering calculated over the frequency range of 3.2-6.4 MHz using various receive transducers and Equation (1).