The dance of the nanobubbles: detecting acoustic backscatter from sub-micron bubbles using ultra-high frequency acoustic microscopy.

Nanobubbles have gained attention for their use as highly stable ultrasound (US) contrast agents, but assessment of individual nanobubble size remains a challenge. Current sizing techniques require either extensive sample preparation or depend on assumed values of nanobubble density that are not well characterized. An US based approach would be desirable; however, probing individual nanobubbles using US transducers at clinical frequencies is not feasible due to the comparatively long acoustic wavelengths employed. Here we present a technique which can be used to estimate nano- or microbubble size by virtue of the amount of motion detected in an M-Mode image acquired using an acoustic microscope equipped with a 200 MHz transducer. A sample of a bubble-containing solution is incorporated into a phantom composed of molten agarose. The solidified agarose gel contains pores with well-defined sizes dictated by the agarose concentration. Bubbles in the gel matrix that are smaller in diameter than the gel pore size are capable of undergoing stochastic motion which manifests as intensity fluctuations in M-Mode images. Conversely, bubbles which are larger than the agarose pores become trapped and produce static M-Mode intensity patterns. In this study, agarose gels with concentrations ranging from 0.25% to 1.25% (mean pore sizes ranging from 2.68 μm to 0.34 μm) were loaded with either nanobubbles (mean diameter 0.326 μm) or microbubbles (mean diameter 2.71 μm) and imaged at 200 MHz. In the nanobubble loaded gels, M-Mode fluctuations were clearly visible up to a gel concentration of 1% (pore size of 0.39 μm). In contrast, the microbubble loaded gels exhibited minimal M-Mode fluctuation even at agarose concentrations of 0.25% (2.68 μm pore size). Autocorrelation curves generated from the M-Mode data demonstrated a clear trend of curve flattening (loss of motion) when the pore size was comparable to mean bubble diameter, indicating that individual bubbles trapped in the agarose pores are the main source of acoustic backscatter. In the future, decay parameters extracted from the autocorrelation curves could potentially be used as indicators of mean bubble diameter for bubble populations of unknown size.