Figure 4. Gas vesicles produce ultrasound contrast in vivo.

a, Overlay of second harmonic image (6 MHz pulses) in green on a grayscale broadband anatomical image of mouse lower abdomen injected subcutaneously with 150 µL OD 6.0 Halo GVs on the right side and 150uL PBS on the left side. b–c, Second harmonic ultrasound image before (b) and after (c) GV collapse with destructive insonation (650 kPa). Dashed outlines show regions of interest (ROI) used to quantify signals. d. Total back-scatted second harmonic signal from ROIs covering GV-injected (orange) and PBS-injected (blue) tissues, before and after collapse (N=5). e–g. Non-linear contrast images acquired using a high-frequency ultrasound scanner system (operating at 18 MHz and 2% power) of SCID nude mice infused intravenously with 50 µL OD 6.0 Halo GVs. The images show contrast at 4.5 seconds (e) and 64 seconds (f) after the start of infusion, or after the application of a burst pulse (g). The locations of the inferior vena cava (IVC) and liver are indicated with labels. h. Time course of the smoothed average non-linear signal in the IVC (blue) and liver (orange) during infusion. i. Mean average signal intensity in the IVC before (pre) during (peak) and after (steady) infusion, and after the burst pulse (post) (N=5). j. Mean average signal intensity in the liver before (pre) and after (steady) infusion, and after the burst pulse (post) (N=5). k–l. Dose-response relationship of 50 µL Halo GVs infused at OD 0 – 6.0 determined from the area under the curve (AUC) of average contrast in the IVC (k) and liver (l) (N=5). Detailed image acquisition and analysis parameters are provided in Supplementary Table S1; colour maps for ultrasound images in Supplementary Fig. S9. The size of each field of view is indicated in the lower right corner of the image. All error bars represent ± SEM.