Abstract-The structure of microvasculature cannot be resolved using standard clinical ultrasound (US) imaging frequencies due to the fundamental diffraction limit of US waves. In this work, we use a standard clinical US system to perform in vivo sub-diffraction imaging on a CD1, female mouse aged 8 weeks by localizing isolated US signals from bubbles flowing within the ear microvasculature, and compare our results to optical microscopy. Furthermore, we develop a new technique to map blood velocity at super-resolution by tracking individual bubbles through the vasculature. Resolution is improved from a measured lateral and axial resolution of 112 µm and 94 µm respectively in original US data, to super-resolved images of microvasculature where vessel features as fine as 19 µm are clearly visualized. Velocity maps clearly distinguish opposing flow direction and separated speed distributions in adjacent vessels, thereby enabling further differentiation between vessels otherwise not spatially separated in the image. This technique overcomes the diffraction limit to provide a non-invasive means of imaging the microvasculature at super-resolution, to depths of many centimeters. In the future, this method could noninvasively image pathological or therapeutic changes in the microvasculature at centimeter depths in vivo.

The majority of exchanges of oxygen and nutrients are performed around vessels smaller than 100 mm, allowing cells to thrive everywhere in the body. Pathologies such as cancer, diabetes and arteriosclerosis can profoundly alter the microvasculature. Unfortunately, medical imaging modalities only provide indirect observation at this scale. Inspired by optical microscopy, ultrasound localization microscopy has bypassed the classic compromise between penetration and resolution in ultrasonic imaging. By localization of individual injected microbubbles and tracking of their displacement with a subwavelength resolution, vascular and velocity maps can be produced at the scale of the micrometer. Super-resolution ultrasound has also been performed through signal fluctuations with the same type of contrast agents, or through switching on and off nano-sized phase-change contrast agents. These techniques are now being applied pre-clinically and clinically for imaging of the microvasculature of the brain, kidney, skin, tumors and lymph nodes.

Ultrasound (US) is a widely used clinical imaging modality that offers penetration depths in tissue of >10 cm. However, the spatial resolution in US imaging is fundamentally limited by diffraction to approximately half the wavelength of the sound wave employed. The spatial resolution of optical microscopy is limited by the same fundamental physics, but in recent years super-resolution imaging techniques have been developed that overcome the diffraction limit through the localization of many spatially separated photo-switchable or photo-activatable fluorophores. In this paper, we apply a related approach to demonstrate super-resolution imaging with US. We imaged dilute suspensions of microbubble contrast agents flowing through narrow tube-based phantoms. By spatially localizing multiple spatially isolated microbubbles, we constructed super-resolved microbubble location density maps that clearly resolve features 5.1-2.2 times smaller than the US system point spread function full width half maximum in the lateral and axial directions respectively. Our initial characterization experiment using a fixed 100 µm diameter brass wire and a US frequency of 2 MHz suggests that for an ideal stationary point scatterer the ultimate resolution of the unmodified clinical US system used could be in the range of 2-4 µm.

Encapsulated microbubbles are well established as highly effective contrast agents for ultrasound imaging. There remain, however, some significant challenges to fully realize the potential of microbubbles in advanced applications such as perfusion mapping, targeted drug delivery, and gene therapy. A key requirement is accurate characterization of the viscoelastic surface properties of the microbubbles, but methods for independent, nondestructive quantification and mapping of these properties are currently lacking. We present here a strategy for performing these measurements that uses a small fluorophore termed a "molecular rotor" embedded in the microbubble surface, whose fluorescence lifetime is directly related to the viscosity of its surroundings. We apply fluorescence lifetime imaging to show that shell viscosities vary widely across the population of the microbubbles and are influenced by the shell composition and the manufacturing process. We also demonstrate that heterogeneous viscosity distributions exist within individual microbubble shells even with a single surfactant component.

Ultrasound-mediated optical tomography (UOT) is a hybrid technique that is able to combine the high penetration depth and high spatial resolution of ultrasound imaging to overcome the limits imposed by optical scattering for deep tissue optical sensing and imaging. It has been proposed as a method to detect blood concentrations, oxygenation and metabolism at depth in tissue for the detection of vascularized tumours or the presence of absorbing or scattering contrast agents. In this paper, the basic principles of the method are outlined and methods for simulating the UOT signal are described. The main detection methods are then summarized with a discussion of the advantages and disadvantages of each. The recent focus on increasing the weak UOT signal through the use of the acoustic radiation force is explained, together with a summary of our results showing sensitivity to the mechanical shear stiffness and optical absorption properties of tissue-mimicking phantoms.

Magnetic resonance imaging and X-ray computed tomography provide the two principal methods available for imaging the brain at high spatial resolution, but these methods are not easily portable and cannot be applied safely to all patients. Ultrasound imaging is portable and universally safe, but existing modalities cannot image usefully inside the adult human skull. We use in silico simulations to demonstrate that full-waveform inversion, a computational technique originally developed in geophysics, is able to generate accurate three-dimensional images of the brain with sub-millimetre resolution. This approach overcomes the familiar problems of conventional ultrasound neuroimaging by using the following: transcranial ultrasound that is not obscured by strong reflections from the skull, low frequencies that are readily transmitted with good signal-to-noise ratio, an accurate wave equation that properly accounts for the physics of wave propagation, and adaptive waveform inversion that is able to create an accurate model of the skull that then compensates properly for wavefront distortion. Laboratory ultrasound data, using ex vivo human skulls and in vivo transcranial signals, demonstrate that our computational experiments mimic the penetration and signal-to-noise ratios expected in clinical applications. This form of non-invasive neuroimaging has the potential for the rapid diagnosis of stroke and head trauma, and for the provision of routine monitoring of a wide range of neurological conditions.

No abstract

The high efficiency with which gas microbubbles can scatter ultrasound compared with the surrounding blood pool or tissues has led to their widespread employment as contrast agents in ultrasound imaging. In recent years, their applications have been extended to include super-resolution imaging and the stimulation of localized bio-effects for therapy. The growing exploitation of contrast agents in ultrasound and in particular these recent developments have amplified the need to characterize and fully understand microbubble behavior. The aim in doing so is to more fully exploit their utility for both diagnostic imaging and potential future therapeutic applications. This paper presents the key characteristics of microbubbles that determine their efficacy in diagnostic and therapeutic applications and the corresponding techniques for their measurement. In each case, we have presented information regarding the methods available and their respective strengths and limitations, with the aim of presenting information relevant to the selection of appropriate characterization methods. First, we examine methods for determining the physical properties of microbubble suspensions and then techniques for acoustic characterization of both suspensions and single microbubbles. The next section covers characterization of microbubbles as therapeutic agents, including as drug carriers for which detailed understanding of their surface characteristics and drug loading capacity is required. Finally, we discuss the attempts that have been made to allow comparison across the methods employed by various groups to characterize and describe their microbubble suspensions and promote wider discussion and comparison of microbubble behavior.