**1. Introduction**

Ultrasound (US) imaging, also known as sonography, is used routinely in clinics for examining various organs of the body to diagnose, localize, and characterize diseases. Its advantages are low costs, real-time imaging capability, and the lack of exposure of the patient to radioactive rays. During US imaging the body is exposed to high-frequency sound waves, which are reflected by the tissues. The US probe is detecting the echoes and by calculating the amplitude and time of the reflected waves, an image is generated. Since tissue penetration decreases with increasing frequency of the sound waves but resolution increases, harmonic imaging was developed, which considers not only the center frequency of the echoes, but also their higher harmonics [1].

US contrast agents (UCA) can be used to visualize the vasculature of the tissue. The standard UCA are small gas-filled spheres, mainly referred to as microbubbles (MB), which are stabilized by a shell layer formed of lipids, proteins or polymers [2]. MB typically have a size between 1 and 10 μm. Therefore, they do not leave the vasculature. Due to the acoustic impedance difference between gas and blood, UCA can be easily distinguished from the tissue. Hereby, an increase in the signal to noise ratio is achieved.

Furthermore, decorating UCA with targeting moieties, specific disease markers can be detected and quantified. Various intravascular targets have been proposed for MB targeting such as integrins, selectins, and cell adhesion molecules. The preclinical results indicate that actively targeted MB can detect angiogenesis, inflammation, and thrombus formation [3]. In this context, even small changes in marker expression can be quantified by molecular US. Undoubtfully, molecular US imaging has high potential for clinical translation, and two targeted MB formulations are already in clinical trials. Complementary to MB, there is increasing research activity on molecular US imaging using nanobubbles (NB) and their targeting to extravascular markers [4]. UCA are also employed for US mediated drug and gene delivery. Drug or gene uptake is facilitiated by increasing the permeability of cell membranes and biological barriers with oscillating or bursting MB. However, this topic is already addressed in recent review articles [5–7].

Therefore, this review article focus on the evolution of molecular US, highlighting the newest molecular US imaging technologies, providing an overview of targeted contrast agent formulations, and summarizing their applications in preclinical and clinical studies. In the context of the latter, this paper also discusses challenges that have to be overcome to accelerate clinical translation.
