# An Open Access Chamber Designed for the Acoustic Characterisation of Microbubbles
## 2 Materials and Methods
### Experiment Design  
The MACC design has been used to measure the acoustic scattering at the fundamental frequency and second harmonic of SonoVue(r) microbubbles. The experiment was performed with a 16-channel variant of the Ultrasound Array Research Platform (UARPII-16) developed at the University of Leeds and shown in Figure 5. This open platform allows the connection of 16 single-element transducers and can transmit and receive on all 16 channels simultaneously. The MACC, UARPII-16, and all six unfocused transducers were used to perform an experiment to assess the suitability of the entire setup for microbubble acoustic measurements [33]. Whilst the UARPII-16 was used in this instance, it could have been replaced with another multi-channel system providing access to the radio-frequency data from every channel [34] such as the NDT Vantage(r) (Verasonics(r), Kirkland, WA, USA) research solutions. Alternatively, a setup consisting of an arbitrary waveform generator, power amplifier, and oscilloscope [7,27,29] could be used, provided this setup accommodates six channels. The UARPII-16 uses a quinary switched excitation method [35] to excite the transducers. This method allows the synthesis of an arbitrary waveform. Narrowband pulses, with a 10 us duration, were used as excitation waveforms. The waveforms were stepped in frequency ranging from 1.6 to 6.4 MHz at increments of 0.2 MHz. Only the waveforms that fell within the 60% fractional bandwidth of a transducer were used with that transducer. This ensured that the frequency measurements from the various transducers overlapped whilst operating within reasonable limits of the transducer bandwidth. The waveforms were windowed using a Tukey window with a 0.2 Cosine fraction. The transducer response to these waveforms was measured before the experiment using a membrane hydrophone (Precision Acoustics, Dorset, UK) in a large water tank filled with deionised water. The membrane hydrophone was positioned to record the maximum pressure at a distance of 35 mm from the transducer under test. An oscilloscope (MSO-S 204 A, Keysight Technologies, Berkshire, UK) was used to obtain 128 acquisitions, and these were subsequently averaged in the time-domain. The resulting waveforms were windowed to obtain the central 5 us of the received pulse and inverse filtered using the hydrophone frequency response to obtain the pressure value corresponding to the maximum amplitude in the frequency domain. The amplitude of the transmit waveform was then pre-distorted to obtain a uniform 70 kPa root mean square (RMS) pressure at the transmit frequency used for each transducer. This was validated through further measurements with the membrane hydrophone. The chamber was populated with all six transducers, and the pairs of transducers corresponding to the same central frequency were placed in line with each other according to Figure 4. The chamber was filled with deionised water. Due to the geometry of the chamber, it is possible for air to accumulate and become trapped within the upper two transducer housings. To prevent this from happening, the outlet stopcock was closed, and the upper transducer connectors were loosened during the initial filling stage to allow air to escape through the top of the upper transducer housings. The connectors were then tightened once more, and the outlet stopcock was opened for experimental measurements. A total of 110 mL of water was required to fill the chamber. Flow rates were assessed before the experiment when characterising the chamber. During this assessment, dye was applied to the sample to visualise the flow-focusing behaviour. A sheath flow rate of 22 mL per minute and a sample flow rate of 7 mL per minute were determined to provide a stable sample stream. This resulted in the sample being constrained to a stream approximately 1 mm in diameter in the centre of the chamber. It is worth noting that this process should be repeated for individually manufactured inlet housings to account for manufacturing discrepancies.