Quantifying activation of perfluorocarbon-based phase-change contrast agents using simultaneous acoustic and optical observation

Abstract

Phase-change contrast agents in the form of nanoscale droplets can be activated into microbubbles by ultrasound, extending the contrast beyond the vasculature. This article describes simultaneous optical and acoustical measurements for quantifying the ultrasound activation of phase-change contrast agents over a range of concentrations. In experiments, decafluorobutane-based nanodroplets of different dilutions were sonicated with a high-pressure activation pulse and two low-pressure interrogation pulses immediately before and after the activation pulse. The differences between the pre- and post-interrogation signals were calculated to quantify the acoustic power scattered by the microbubbles activated over a range of droplet concentrations. Optical observation occurred simultaneously with the acoustic measurement, and the pre- and post-microscopy images were processed to generate an independent quantitative indicator of the activated microbubble concentration. Both optical and acoustic measurements revealed linear relationships to the droplet concentration at a low concentration range <10(8)/mL when measured at body temperature. Further increases in droplet concentration resulted in saturation of the acoustic interrogation signal. Compared with body temperature, room temperature was found to produce much fewer and larger bubbles after ultrasound droplet activation.

Keywords: Acoustic droplet vaporization; Concentration; Contrast agent; Microbubble; Perfluorocarbon droplet; Phase change; Quantification; Temperature.

Fig. 1

Microscopic images focused at the top and bottom planes of the cytometer containing microbubbles only (a, c) and droplets with few spontaneously activated bubbles (b, d). Bar = 20 μm.

Fig. 2

Schematic of experimental setup.

Fig. 3

Procedure for optical quantification of acoustic droplet activation (a–d). Bar = 20 μm.

Fig. 4

Microscopic images (a–f) and corresponding interrogation echo signals (g–l) acquired before and after the “activation pulse” in droplets and controls. Bar = 20 μm.

Fig. 5

Power spectral density (PSD) of the “difference signal” detected with droplets and control.

Fig. 6

(a–d) Microscopic images of variously diluted droplet emulsions acquired after droplet vaporization. Bar = 20 μm. (e) Optical quantification as an indicator of the concentration of generated bubbles. Data at relative concentrations >0.04 (stars) were not included in the linear fitting.

Fig. 7

Acoustic measurement of droplet vaporization: (a) power spectral density and (b) power of the interrogation “difference signal.” Data at relative concentrations 0–2% (diamond markers) were used in the linear fitting, as illustrated in the inset in (b).

Fig. 8

Microscopic images of the acoustically vaporized droplets at different temperatures. (a) Undiluted droplets activated at 21°C. (b) Droplets diluted to 1:3 and activated at 37°C. Bar = 20 μm.

Fig. 9

(a) Acoustic and (b) optical quantification of droplet activation at 21°C. Both measurements (50 repetitions) were normalized to the results measured using the droplets with a relative concentration of 0.2 (20%).