Effect of microbubble size on fundamental mode high frequency ultrasound imaging in mice

Abstract

High-frequency ultrasound imaging using microbubble (MB) contrast agents is becoming increasingly popular in pre-clinical and small animal studies of anatomy, flow and vascular expression of molecular epitopes. Currently, in vivo imaging studies rely on highly polydisperse microbubble suspensions, which may provide a complex and varied acoustic response. To study the effect of individual microbubble size populations, microbubbles of 1-2 microm, 4-5 microm and 6-8 microm diameter were isolated using the technique of differential centrifugation. Size-selected microbubbles were imaged in the mouse kidney over a range of concentrations using a Visualsonics Vevo 770 ultrasound imaging system (Visualsonics, Toronto, Ontario, Canada) with a 40-MHz probe in fundamental mode. Results demonstrate that contrast enhancement and circulation persistence are strongly dependent on microbubble size and concentration. Large microbubbles (4-5 and 6-8 microm) strongly enhanced the ultrasound image with positive contrast, while 1-2 microm microbubbles showed little enhancement. For example, the total integrated contrast enhancement, measured by the area under the time-intensity curve (AUC), increased 16-fold for 6-8 microm diameter microbubbles at 5 x 10(7) MB/bolus compared with 4-5 microm microbubbles at the same concentration. Interestingly, 1-2 microm diameter microbubbles, at any concentration, did not measurably enhance the integrated ultrasound signal at tissue depth, but did noticeably attenuate the signal, indicating that they had a low scattering-to-attenuation ratio. When concentration matched, larger microbubbles were more persistent in circulation. However, when volume matched, all microbubble sizes had a similar circulation half-life. These results indicated that dissolution of the gas core plays a larger role in contrast elimination than filtering by the lungs and spleen. The results of this study show that microbubbles can be tailored for optimal contrast enhancement in fundamental mode imaging.

Figure 1

Size distributions of freshly made microbubble suspensions. Microbubbles were formulated using a Vialmix shaker for small volumes of lipid solution (2 mL) and probe sonication for large volumes of lipid solution (120 mL). All lipid solutions (2 mg-lipid/mL) consisted of DSPC and PEG40S at a 9:1 molar ratio.

Fig. 2

Size distributions of size-selected microbubbles. The microbubble populations for the individual 1-2 μm (blue), 4-5 μm (red), and 6-8 μm (green), samples are shown as number-weighted and volume-weighted size distributions. The polydisperse sample (grey) is from a freshly made microbubble suspension generated using a shaker.

Figure 3

Contrast enhancement in the kidney following bolus injections of size-selected microbubbles. Microbubble suspensions of 100 μL containing 5×10⁷ MB of (A) polydisperse, (B) 1-2 μm, (C) 4-5 μm, or (D) 6-8 μm diameter bubbles were injected intravenously into anesthetized mice while continuously imaging the kidney using a 40-MHz ultrasound probe. Grayscale images are shown before the bolus is injected (A1-D1) and at the peak signal intensity (A2-D2), typically 30-60 seconds after the bolus was delivered. Contrast detection software was used to highlight the presence of microbubbles in green (A3-D3). Areas outlined in white are ROIs selected for TIC analysis.

Figure 4

Attenuation of the ultrasound signal from 1-2 μm bubbles. (A) The change in video intensity caused by the 1-2 μm bubbles was evaluated over two regions of interest. Typically, a smaller region of interest in the upper portion of the kidney (yellow) was used to avoid effects of attenuation and shadowing. The larger region of interest (green) encompassed areas deeper within the tissue, which were prone to shadowing and attenuation by microbubbles. (B) The relative acoustic backscatter of the ultrasound signal from the 1-2 μm bubbles showed little signal enhancement above noise in the smaller region of interest. (C) The 1-2 μm bubbles showed strong attenuation of the signal, as determined by whole kidney as the region of interest.

Figure 5

Time-intensity curves in the mouse kidney following bolus injections of size-selected microbubble suspensions. Representative TIC’s are shown for each size-selected population after a 100 μL bolus injection of 5×10⁷ MB. All data was offset corrected using background images as a reference.

Figure 6

Signal amplitude and half-life of size-selected microbubbles. The signal amplitude and total contrast persistence was measured from the TIC’s over a range of microbubble concentrations. (A) The signal amplitude was determined by the maximum signal intensity from the TIC for each microbubble sample. For 1-2 μm bubbles which attenuated the signal, the minimum signal intensity value was used. (B) The half-life of the signal was determined from the TIC at the time the signal intensity decayed to half of the maximum amplitude.

Figure 7

Signal amplitude and half-life of size-selected microbubbles as a function of total gas volume. The signal amplitude values (A) and half-life values (B) are the same as shown in Figure 6.

Figure 8

Parameters for model fit of TIC. Each TIC was fit to a model describing the influx and decay of contrast agent in the kidney (equation 1 in Appendix). The parameters were determined by fitting the data to the equation using least a squares regression analysis. (A) D_o is proportional to the signal intensity. (B) k₁ is a measure of the influx rate of the contrast agent into the kidney. (C) k₂ is a measure of the decay rate of the ultrasound contrast signal. (D) AUC is a measure of the total integrated contrast enhancement determined by the area under the fitted curve.

Figure 9

(A) Extinction cross-section as a function of diameter. The extinction cross section, absorption cross section and scattering cross section were calculated using equations derived by Medwin (Medwin 1977) with an additional damping coefficient due to frictional loss (de Jong et al. 1992; de Jong and Hoff 1993) (B) The effect of surface tension on extinction cross-section. The extinction-crossection as a function of diameter was plotted for low (0 mN/m) and high (72 mN/m) surface tensions to determine the effect of rapidly changing surface tension on the microbubble shell in an ultrasound field. Grey area represents surface tensions between 0 and 72 mN/m.