  Among the various molecular imaging modalities, ultrasound imaging benefits
from its real-time, nonionizing, and cost-effective nature. Despite its
benefits, there is a dearth of methods to visualize two or more populations of
contrast agents simultaneously, a technique known as multiplex imaging. In this
paper, we present a new approach to multiplex ultrasound imaging using
perfluorocarbon (PFC) nanodroplets. The nanodroplets, which undergo a
liquid-to-gas phase transition in response to an acoustic trigger, act as
activatable contrast agents. By using two populations of PFC nanodroplets, each
with a different core boiling point, their unique temporal responses to an
acoustic trigger were leveraged to differentiate their unique contributions to
the overall ultrasound signal. This work characterized the dynamic responses of
two PFC nanodroplets with boiling points of 28 and 56 {\deg}C. These
characteristic responses were then used to demonstrate that the relative
concentrations of the two populations of PFC nanodroplets could be accurately
measured in the same imaging volume within an average error of 1.1%. Overall,
the findings indicate the potential of this approach for multiplex ultrasound
imaging, allowing for the visualization of multiple molecular targets
simultaneously.
