Laser-activated perfluorocarbon nanodroplets (PFCnDs) are emerging phase-change contrast agents that showed promising potential in ultrasound and photoacoustic (US/PA) imaging. Unlike monophase gaseous microbubbles, PFCnDs shift their state from liquid to gas via optical activation and can provide high US/PA contrast on demand. Depending on the choice of perfluorocarbon core, the vaporization and condensation dynamics of the PFCnDs are controllable.
Over the past decade, phase-change contrast agents were rigorously studied and showed promising outcomes in diagnostic ultrasound imaging and therapeutic applications . These contrast agents consisting of a liquid perfluorocarbon (PFC) core, referred to as perfluorocarbon nanodroplets (PFCnDs), undergo a phase transition from liquid to gaseous state in response to an external trigger . Ultrasound energy (or optical energy for laser activation of PFCnDs with a photo-absorber) can be applied to induce vaporization of PFCnDs, capable of providing on-demand controllable contrast. Unlike micrometer-sized gaseous bubbles, these liquid PFCnDs can be stably generated in a broad range of sizes down to a few hundred nanometers and remain stable in blood circulation . Thus, applications of the PFCnDs are not necessarily limited to the vascular space . Before activation, the administered PFCnDs could be small enough to extravasate through the leaky cancerous neovasculature for extravascular cancer imaging.
2. Activation and Deactivation of Laser-Activated PFCnDs
Activation and deactivation (in other words, vaporization and recondensation) of the laser-activated PFCnDs are controlled by a combined function of many factors, including perfluorocarbon type, droplet size, local optical fluence, and amount of dye encapsulated 
. A boiling point of perfluorocarbon core relative to surrounding physiological temperature (37 °C) is one of critical factors on both activation and deactivation of the PFCnDs. Higher optical fluence is generally needed to activate PFCnDs with a higher boiling point PFC.
Table 1. Boiling points of selected PFCs user for PFCnDs.
|Perfluorocarbon Name (Chemical Formular)
||Boiling Point (°C)
For optical activation, pulsed-laser irradiation enables vaporization of laser-activated PFCnDs. The suspended plasmonic nanoparticles or dyes encapsulated in the droplet generate the heat and pressure, leading to vaporization of the PFC liquid core 
. Wilson et al. showed that photoacoustic signals from vaporization comes first and subsequent thermal expansion is followed over a course of laser pulses 
. Here, their PFCnDs are constructed with a low boiling point PFC (e.g., perfluoropentane in this study), only one-time activation is available at the beginning of laser irradiation, producing high-contrast photoacoustic signals. Relatively weaker photoacoustic signals are then generated during a series of following thermal expansion.
Activating deep and/or small PFCnDs is, however, challenging, which may require the optical fluence exceeding the American National Standards Institute safety limit for successful activation. Thus, to lower the optical activation threshold, Arnal et al. suggested sono-photoacoustic (SPA) imaging that synchronously combines acoustic and laser pulses 
. To have both acoustic and laser pulses at the depth of interest and at the same time, the acoustic pulse is sent first as it is much slower than the laser pulse. Depending on the depth of interest, a traveling depth can be precalculated, and thus, the time delay between acoustic and laser pulses can be determined. With this approach, they were able to synchronize acoustic and optical activation pulses, which substantially lowered the PFCnD activation threshold. In addition, to cancel out unwanted linear photoacoustic sources and acoustic scatters, they specifically designed an imaging sequence with four successive ultrasound transmissions with alternating polarities. Similar concept was utilized for inertial cavitation-based sonoporation for tumor therapy 
. More recently, Li et al. improved the SPA imaging sequence by applying focused and steered ultrasound beams, which enables highly localized activation of PFCnDs 
Perfluorohexane and perfluoropentane are commonly used materials for laser-activated PFCnDs. Compared to that of their similar vaporization properties, their recondensation behaviors are indeed more different 
. A boiling point of perfluorohexane is higher than a body temperature, and thus, perfluorohexane nanodroplets (PFHnDs) can condense back to their original liquid form after vaporization, as shown in Figure 1
. However, perfluoropentane nanodroplets (PFPnDs) could remain as gaseous bubbles. Therefore, PFHnDs can repeatedly vaporize and recondense, providing repeated strong photoacoustic contrast and uniquely enabling novel ultrasound imaging methods.
Vaporization and recondensation of laser-activated PFHnDs. Postactivated PFHnDs are imaged with ultrasound and exhibit repeated decaying ultrasound signals over period. Reprinted with permission from 
PFHnD’s recondensation time is, however, relatively transient, ranging from several to hundreds of milliseconds 
. Zhu et al. found that the phase of ultrasound imaging pulse impacts the recondensation dynamics 
. For example, if the initial part of transmit pulse is rarefactional rather than compressional, it was experimentally shown that the corresponding recondensation time is extended, and the signal-to-noise ratio is improved as well. Another important property of droplet recondensation is its stochasticity. As noted earlier, the recondensation process is complex and a combined function of droplet size, local laser fluence, amount of dye, shell composition, and imaging conditions, along with local temperature, pressure, and viscoelasticity. Therefore, isolation of randomly recondensing individual droplet is feasible, which enabled super-resolution imaging 
3. Ultrasound and Photoacoustic Imaging of Laser-Activated PFCnDs
Laser-activated PFCnDs are a dual-modality agent offering both ultrasonic and photoacoustic contrast 
. Most US/PA imaging studies with laser-activated PFCnDs utilized two types of PFCnDs: PFPnDs and PFHnDs. Both as liquid nanodroplets are not easily ultrasonically detectable before optical activation. In response to laser irradiation, the encapsulated dyes or nanoparticles absorb optical energy, producing heat and photoacoustic pressures. This process eventually initiates a liquid-to-gas transition of PFCnDs, resulting in vaporization or volumetric expansion of the droplets. For PFPnDs, after vaporization, they persist their gaseous state. Here, three contrast mechanisms can be appreciated with laser activation of PFPnDs. Volumetric expansion and thermal expansion produce photoacoustic signals, and vaporized gas-filled bubbles can provide high-contrast in ultrasound. As shown in Figure 2
, the first vaporization event yields the strongest photoacoustic signal, and the following thermal expansion results in relatively weaker photoacoustic signals over a course of laser pulses.
In vivo US/PA imaging experiments demonstrating vaporization and thermal expansion-induced photoacoustic signals over a course of laser pulses. Reprinted under permission from 
Exploiting higher-boiling-point PFCnDs (e.g., PFHnDs) offers an ability to reactivate the agent because of their recondensation after vaporization, which enables extended imaging methods, such as background-free contrast-enhanced ultrasound and super-resolution ultrasound imaging 
. A lifetime of vaporized PFHnDs is known to range from several to hundreds of milliseconds, which is relatively transient to utilize them for prolonged high-contrast imaging. To localize the PFHnDs, autocorrelation of a set of ultrasound images containing several events of droplet vaporization and recondensation was suggested. Later, Yoon et al. proposed a computationally-efficient PFHnD-localization algorithm for real-time in vivo imaging 
. By incorporating the periodic nature of droplet vaporization and recondensation in response to laser irradiation, they developed a formula that can map a probability of droplet existence on the image and showed successful differentiation of lymph node of a mouse model from surrounding tissue, as shown in Figure 3
. From the temporal ultrasound profile measured on a pixel containing PFHnDs, vaporization and recondensation behaviors are observable, but not as much clear as phantom imaging in their study 
In vivo contrast-enhanced ultrasound imaging of murine lymph node processed with a PFHnD-localization algorithm over time. Laser pulses start activating the PFHnDs at 0.3 s. Reprinted under permission from 
Compared to that of microbubble-based ultrasound super-resolution imaging, laser-activated PFCnD-based super-resolution methods are relatively new. Luke et al. found that the recondensation of PFHnDs is a stochastic process 
. Depending on many factors, including droplet size, local fluence, and absorbance of dye, the recondensation time of PFHnDs varies randomly. This randomized recondensation of PFHnDs offers a chance to isolate individual droplet if imaging rate is fast enough to capture each recondensation event. Their super-resolution approach shows the 8-fold improvement in spatial resolution over conventional ultrasound. Later, Yoon et al. applied ultrafast plane-wave ultrasound to the super-resolution imaging technique to increase the acquisition frame rate, and also to enhance the image signal-to-noise ratio through temporal compounding 
. They demonstrated that the resolution improves as a function of the number of frames compounded.
US/PA imaging of laser-activated PFHnDs necessitates integrated laser and ultrasound systems and relevant imaging sequences to reach their full potential. Combined multiwavelength photoacoustic and plane-wave ultrasound imaging was suggested to support imaging of transient phase-change contrast agents 
. As a result, US/PA signals from the PFHnDs were measured as a function of both optical wavelength and time, capable of demonstrating the optical wavelength dependency of US/PA imaging and temporal dynamics of PFHnDs simultaneously. However, optical wavelength tuning is too slow to produce real-time images with their approach. Recently, Jeng et al. achieved 50-Hz US/PA imaging rate with sequential spectroscopic laser irradiation 
. Although their system was not demonstrated with phase-change contrast agents, their high-laser-pulse repetition rate should be beneficial to in vivo imaging.
As conventional microbubbles (typically 1–10 μm diameter) cannot leave the blood vessels, they are unable to target or visualize extravascular pathology outside of the vascular space 
. However, due to the submicrometer size of PFCnDs, the PFCnDs can extravasate from leaky cancerous neovasculature and accumulate in tumors by the enhanced permeability and retention (EPR) effect. As a result, they can provide an opportunity for extravascular US/PA imaging of cancer. To achieve reliable extravascular imaging, producing consistently small, monodisperse PFCnDs is critical. Paproski et al. produced the droplets smaller than 200 nm with all organic materials and showed successful accumulation of the droplets in their tumor model 
. Additionally, US/PA imaging was able to identify their nanodroplets specifically. Yarmoska et al. demonstrated that increased molar percentage of lipid reduces the size and size variance of PFCnDs 
. With their nanodroplets, they found substantial photoacoustic contrast enhancement within the primary tumor region of the mouse after 24-h post injection.
In vivo three-dimensional ultrasound and photoacoustic imaging of porphyrin nanodroplets. Yellow, red, and blue photoacoustic signals represent porphyrin nanodroplets, oxygenated hemoglobin, and deoxygenated hemoglobin, respectively. Green scale bar represents 3-mm length. Reprinted under permission from 
Sono-photoacoustic imaging synchronously combines acoustic and optical triggers to lower the overall activation threshold of PFCnDs, and thus, enhances the sensitivity for deeper imaging with smaller agents 
. To obtain background-free droplet-localized images, the authors used a pulse inversion technique for acoustic pulses applied for droplet activation. Thus, with their SPA imaging sequence, specific detection of nanodroplets is possible under low concentration. Later, SPA imaging becomes highly localized with the addition of acoustic beam focusing/steering 
. As demonstrated in Figure 5
, complex patterns can be programmed and drawn with the SPA imaging sequence under acoustic and optical safety limits.
Demonstrated results of steered, localized SPA activation along an arbitrary complex path to draw a letter ‘W’. Reprinted under permission from