Ultrahigh-speed Microscopy Research Articles

Ultrafast dynamics of acoustic vaporization and payload release of phase-shift emulsions

Acoustic droplet vaporization (ADV) is the phase-transitioning of perfluorocarbon emulsions via focused ultrasound. ADV has been utilized in many biomedical applications. For drug delivery and tissue regeneration, we developed composite hydrogel scaffolds, termed acoustically responsive scaffolds (ARSs), which here contained fluorescently labeled monodispersed emulsions (Ø: 12.3 ± 0.8 μm). Using ultra-high speed microscopy, we investigated the dynamics of ADV (2.5 MHz, P-: 6 MPa, pulse length: 6μs) in ARSs containing emulsions with perfluoropentane, perfluorohexane, and perfluorooctane. Vaporization and growth dynamics were studied in micro- to millisecond time scales at frame rates up to 10 Mfps. During ADV, the generated bubbles expanded 4.8-, 2.8-, and 2-fold greater than the initial diameter of the perfluoropentane, perfluorohexane, and perfluorooctane emulsions, respectively. Stable bubbles formed from ADV in perfluoropentane and perfluorohexane, though growth rates post-ADV were significantly different. Bubbles recondensed within perfluorooctane emulsions, which exhibited a 20% decrease in diameter following five repeated ADV events. Stable or transient bubble formation was further compared with classical nucleation theory predictions. Additionally, we compared the release dynamics of fluorescently labeled payloads from the emulsions. The results provide physical insight enabling the modulation of bubble dynamics with ADV and hence release kinetics, which can be used for therapeutic applications.

Individual lipid encapsulated microbubble radial oscillations: Effects of fluid viscosity.

Ultrasound-stimulated microbubble dynamics have been shown to be dependent on intrinsic bubble properties, including size and shell characteristics. The effect of the surrounding environment on microbubble response, however, has been less investigated. In particular, microbubble optimization studies are generally conducted in water/saline, characterized by a 1 cP viscosity, for application in the vasculature (i.e., 4 cP). In this study, ultra-high speed microscopy was employed to investigate fluid viscosity effects on phospholipid encapsulated microbubble oscillations at 1 MHz, using a single, eight-cycle pulse at peak negative pressures of 100 and 250 kPa. Microbubble oscillations were shown to be affected by fluid viscosity in a size- and pressure-dependent manner. In general, the oscillation amplitudes exhibited by microbubbles between 3 and 6 μm in 1 cP fluid were larger than in 4 cP fluid, reaching a maximum of 1.7-fold at 100 kPa for microbubbles 3.8 μm in diameter and 1.35-fold at 250 kPa for microbubbles 4.8 μm in diameter. Simulation results were in broad agreement at 250 kPa, however generally underestimated the effect of fluid viscosity at 100 kPa. This is the first experimental demonstration documenting the effects of surrounding fluid viscosity on microbubble oscillations, resulting in behavior not entirely predicted by current microbubble models.

Fluid Viscosity Affects the Fragmentation and Inertial Cavitation Threshold of Lipid-Encapsulated Microbubbles

Ultrasound and microbubble optimization studies for therapeutic applications are often conducted in water/saline, with a fluid viscosity of 1 cP. In an in vivo context, microbubbles are situated in blood, a more viscous fluid (∼4 cP). In this study, ultrahigh-speed microscopy and passive cavitation approaches were employed to investigate the effect of fluid viscosity on microbubble behavior at 1 MHz subject to high pressures (0.25–2 MPa). The propensity for individual microbubble (n = 220) fragmentation was found to significantly decrease in 4-cP fluid compared with 1-cP fluid, despite achieving similar maximum radial excursions. Microbubble populations diluted in 4-cP fluid exhibited decreased wideband emissions (up to 10.2 times), and increasingly distinct harmonic emission peaks (e.g., ultraharmonic) with increasing pressure, compared with those in 1-cP fluid. These results suggest that in vitro studies should consider an evaluation using physiologic viscosity perfusate before transitioning to in vivo evaluations.

High throughput acoustic and optical characterization of microbubbles for optimized contrast ultrasound imaging

Echogenic particles, such as microbubbles and volatile liquid micro-/nanodroplets, have shown considerable potential in a variety of clinical diagnostic and therapeutic applications. The accurate prediction of their response to ultrasound excitation is however extremely challenging, and this has hindered the optimization of techniques such as quantitative ultrasound imaging and targeted drug delivery. Existing characterization techniques, such as ultra-high speed microscopy, provide important insights, but suffer from a number of limitations; most significantly difficulty in obtaining large data sets suitable for statistical analysis and the need to physically constrain the particles, thereby altering their dynamics. Here, a microfluidic system is presented that overcomes these challenges to enable the measurement of single echogenic particle response to ultrasound excitation with a throughput of 20 samples/second and an uncertainty below 7% in the measurements. Demonstration of optimized contrast ultrasound imaging is shown based on the characterization of over 100,000 individual SonoVue® bubbles.

Insights into mechanisms of sonothrombolysis using high speed imaging

Thrombotic arterial occlusion is the major cause of acute cardiovascular syndromes such as stroke and myocardial infarction. Exposing the thrombus to microbubbles in the presence of ultrasound facilitates thrombus disruption, and is thus a potentially powerful therapeutic strategy for thromboembolic diseases. However, optimization of “sonothrombolysis” is constrained by a lack of understanding of underlying mechanisms for blood clot disruption in response to ultrasound-induced microbubble vibrations. We tested the hypothesis that inertial cavitation induces mechanical clot disruption by optically characterizing lipid microbubble interactions with thrombus + ultrasound using an ultra-high speed microscopy imaging system capable of imaging at MHz frame rates. A microscope/acoustic stage was used to hold an experimentally created thrombus and microbubbles, which were insonified (1 MHz) during synchronized high speed imaging. Large amplitude microbubble oscillations in response to an inertial cavitation regime caused thrombus deformation and pitting. These data implicate a direct mechanical effect of oscillating microbubbles on mediating clot disruption.

Microbubble behavior during long tone-burst ultrasound excitation

Ultrasound-microbubble mediated therapies have been investigated to restore perfusion and enhance drug/gene delivery, using both short (~μs) and long (~ms) ultrasound tone-bursts. Microbubble oscillation behavior can be observed with ultra-high speed microscopy; however, due to the high frame rate required and limited number of frames available, microbubble dynamic behavior can only be observed for a few acoustic cycles (~μs). In this report, the fate of microbubbles throughout long tone-burst ultrasound exposures (5 ms) was explored via direct microscopic optical observation in conjunction with passive cavitation detection, over a range of acoustic pressures (0.25, 0.5, 1.0, and 1.5 MPa) and microbubble concentrations (2e6, 2e7, and 2e8 MB/ml). The ultrasound system was triggered from UPMC Cam, the ultra-high speed camera system at the University of Pittsburgh Medical Center, such that microbubble activity at various time points (0–5 ms) within the long tone-burst was captured. Microbubbles first underwent stable or inertial cavitation depending on the acoustic pressure used, and then formed gas-filled clusters that continued to oscillate, break up, and form new clusters. Cavitation detection confirmed continued, albeit diminishing acoustic activity throughout the 5 ms ultrasound excitation. This discovery suggests that persisting cavitation activity during long tone-bursts may confer additional therapeutic effect for ultrasound-microbubble mediated therapies.

A bright and long-pulse illumination for ultrahigh-speed microscopy of living specimens

Ultrahigh-speed microscopy of living specimens requires ultrabright illumination. Moreover, the duration of illumination should be sufficiently long, on the order of at least several tens of milliseconds, in order to investigate the dynamic state of living specimens. However, specimens are exposed to a high risk of damage by the intense illumination. The brightness and pulse duration of illumination have to be continuously controlled for use in the ultrahigh-speed microscopy of living specimens. Commercial or laboratory-made illumination systems do not satisfy the abovementioned requirements. In this paper, the development of a bright and long-pulse illumination system for ultrahigh-speed microscopy of living specimens is presented. A xenon flashlamp with an arc length of 1.5 mm has been used as the light source. The electrical power supply consists of a voltage-regulated circuit, a capacitor bank, and a control circuit including an insulated-gate bipolar transistor as a gating device, which provides a large rectangular current pulse with the duration in the range to the order of several tens of milliseconds. The brightness, pulse duration, and repetition rate can be easily and continuously controlled. The illumination developed in the present study is installed in an inverted fluorescence microscope equipped with a high-speed camera in order to evaluate the performance as an illumination source. A fluorescent image of the living spermatozoa of a mouse obtained at a frame rate of 8 kHz shows good contrast. Such an image cannot be obtained using a commercial illumination system.