3D functional ultrasound imaging of pigeons.

Recent advances in ultrasound Doppler imaging have facilitated the technique of functional ultrasound (fUS) which enables visualization of brain-activity due to neurovascular coupling. As of yet, this technique has been applied to rodents as well as to human subjects during awake craniotomy surgery and human newborns. Here we demonstrate the first successful fUS studies on awake pigeons subjected to auditory and visual stimulation. To allow successful fUS on pigeons we improved the temporal resolution of fUS up to 20,000 frames per second with real-time visualization and continuous recording. We show that this gain in temporal resolution significantly increases the sensitivity for detecting small fluctuations in cerebral blood flow and volume which may reflect increased local neural activity. Through this increased sensitivity we were able to capture the elaborate 3D neural activity pattern evoked by a complex stimulation pattern, such as a moving light source. By pushing the limits of fUS further, we have reaffirmed the enormous potential of this technique as a new standard in functional brain imaging with the capacity to unravel unknown, stimulus related hemodynamics with excellent spatiotemporal resolution with a wide field of view.

Coupling Two Ultra-high-Speed Cameras to Elucidate Ultrasound Contrast-Mediated Imaging and Therapy.

Ultrasound contrast-mediated medical imaging and therapy both rely on the dynamics of micron- and nanometer-sized ultrasound cavitation nuclei, such as phospholipid-coated microbubbles and phase-change droplets. Ultrasound cavitation nuclei respond non-linearly to ultrasound on a nanosecond time scale that necessitates the use of ultra-high-speed imaging to fully visualize these dynamics in detail. In this study, we developed an ultra-high-speed optical imaging system that can record up to 20 million frames per second (Mfps) by coupling two small-sized, commercially available, 10-Mfps cameras. The timing and reliability of the interleaved cameras needed to achieve 20 Mfps was validated using two synchronized light-emitting diode strobe lights. Once verified, ultrasound-activated microbubble responses were recorded and analyzed. A unique characteristic of this coupled system is its ability to be reconfigured to provide orthogonal observations at 10 Mfps. Acoustic droplet vaporization was imaged from two orthogonal views, by which the 3-D dynamics of the phase transition could be visualized. This optical imaging system provides the temporal resolution and experimental flexibility needed to further elucidate the dynamics of ultrasound cavitation nuclei to potentiate the clinical translation of ultrasound-mediated imaging and therapy developments.

Real-Time Coded Excitation Imaging Using a CMUT-Based Side Looking Array for Intravascular Ultrasound.

Intravascular ultrasound (IVUS) is a well-established diagnostic method that provides images of the vessel wall and atherosclerotic plaques. We investigate the potential for phased-array IVUS utilizing coded excitation (CE) for improving the penetration depth and image signal-to-noise ratio (SNR). It is realized on a new experimental broadband capacitive micromachined ultrasound transducer (CMUT) array, operated in collapse mode, with 96 elements placed at the circumference of a catheter tip with a 1.2- mm diameter. We characterized the array performance for CE imaging and showed that the -6-dB device bandwidth at a 30-V dc biasing is 25 MHz with a 20-MHz center frequency, with a transmit sensitivity of 37 kPa/V at that frequency. We designed a linear frequency modulation code to improve penetration depth by compensating for high-frequency attenuation while preserving resolution by a mismatched filter reconstruction. We imaged a wire phantom and a human coronary artery plaque. By assessing the image quality of the reconstructed wire phantom image, we achieved 60- and 70- µm axial resolutions using the short pulse and coded signal, respectively, and gained 8 dB in SNR for CE. Our developed system shows 20-frames/s, pixel-based beam-formed, real-time IVUS images.

Lipid-rich Plaques Detected by Near-infrared Spectroscopy Are More Frequently Exposed to High Shear Stress.

High wall shear stress (WSS) and near-infrared spectroscopy (NIRS) detected lipid-rich plaque (LRP) are both known to be associated with plaque destabilization and future adverse cardiovascular events. However, knowledge of spatial co-localization of LRP and high WSS is lacking. This study investigated the co-localization of LRP based on NIRS and high WSS. Fifty-three patients presenting acute coronary syndrome underwent NIRS-intravascular-ultrasound (NIRS-IVUS) imaging of a non-culprit coronary artery. WSS was obtained using WSS profiling in 3D-reconstructions of the coronary arteries based on fusion of IVUS-segmented lumen and CT-derived 3D-centerline. Thirty-eight vessels were available for final analysis and divided into 0.5 mm/45° sectors. LRP sectors, as identified by NIRS, were more often colocalized with high WSS than sectors without LRP. Moreover, there was a dose-dependent relationship between lipid content and high WSS exposure. This study is a first step in understanding the evolution of LRPs to vulnerable plaques. Graphical Abstract.

Simultaneous Morphological and Flow Imaging Enabled by Megahertz Intravascular Doppler Optical Coherence Tomography.

We demonstrate three-dimensional intravascular flow imaging compatible with routine clinical image acquisition workflow by means of megahertz (MHz) intravascular Doppler Optical Coherence Tomography (OCT). The OCT system relies on a 1.1 mm diameter motorized imaging catheter and a 1.5 MHz Fourier Domain Mode Locked (FDML) laser. Using a post processing method to compensate the drift of the FDML laser output, we can resolve the Doppler phase shift between two adjoining OCT A-line datasets. By interpretation of the velocity field as measured around the zero phase shift, the flow direction at specific angles can be qualitatively estimated. Imaging experiments were carried out in phantoms, micro channels, and swine coronary artery in vitro at a speed of 600 frames/s. The MHz wavelength sweep rate of the OCT system allows us to directly investigate flow velocity of up to 37.5 cm/s while computationally expensive phase-unwrapping has to be applied to measure such high speed using conventional OCT system. The MHz sweep rate also enables a volumetric Doppler imaging even with a fast pullback at 40 mm/s. We present the first simultaneously recorded 3D morphological images and Doppler flow profiles. Flow pattern estimation and three-dimensional structural reconstruction of entire coronary artery are achieved using a single OCT pullback dataset.

High-Resolution Imaging of Intracellular Calcium Fluctuations Caused by Oscillating Microbubbles.

Ultrasound insonification of microbubbles can locally enhance drug delivery, but the microbubble-cell interaction remains poorly understood. Because intracellular calcium (Cai2+) is a key cellular regulator, unraveling the Cai2+ fluctuations caused by an oscillating microbubble provides crucial insight into the underlying bio-effects. Therefore, we developed an optical imaging system at nanometer and nanosecond resolution that can resolve Cai2+ fluctuations and microbubble oscillations. Using this system, we clearly distinguished three Cai2+ uptake profiles upon sonoporation of endothelial cells, which strongly correlated with the microbubble oscillation amplitude, severity of sonoporation and opening of cell-cell contacts. We found a narrow operating range for viable drug delivery without lethal cell damage. Moreover, adjacent cells were affected by a calcium wave propagating at 15 µm/s. With the unique optical system, we unraveled the microbubble oscillation behavior required for drug delivery and Cai2+ fluctuations, providing new insight into the microbubble-cell interaction to aid clinical translation.

Opening of endothelial cell-cell contacts due to sonoporation.

Ultrasound insonification of microbubbles can locally increase vascular permeability to enhance drug delivery. To control and optimize the therapeutic potential, we need to better understand the underlying biological mechanisms of the drug delivery pathways. The aim of this in vitro study was to elucidate the microbubble-endothelial cell interaction using the Brandaris 128 ultra-high-speed camera (up to 25 Mfps) coupled to a custom-built Nikon confocal microscope, to visualize both microbubble oscillation and the cellular response. Sonoporation and opening of cell-cell contacts by single αVß3-targeted microbubbles (n = 152) was monitored up to 4 min after ultrasound insonification (2 MHz, 100-400 kPa, 10 cycles). Sonoporation occurred when microbubble excursion amplitudes exceeded 0.7 µm. Quantification of the influx of the fluorescent model drug propidium iodide upon sonoporation showed that the size of the created pore increased for larger microbubble excursion amplitudes. Microbubble-mediated opening of cell-cell contacts occurred as a cellular response upon sonoporation and did not correlate with the microbubble excursion amplitude itself. The initial integrity of the cell-cell contacts affected the susceptibly to drug delivery, since cell-cell contacts opened more often when cells were only partially attached to their neighbors (48%) than when fully attached (14%). The drug delivery outcomes were independent of nonlinear microbubble behavior, microbubble location, and cell size. In conclusion, by studying the microbubble-cell interaction at nanosecond and nanometer resolution the relationship between drug delivery pathways and their underlying mechanisms was further unraveled. These novel insights will aid the development of safe and efficient microbubble-mediated drug delivery.

Effects of the direct lipoprotein-associated phospholipase A(2) inhibitor darapladib on human coronary atherosclerotic plaque.

BACKGROUND: Lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is expressed abundantly in the necrotic core of coronary lesions, and products of its enzymatic activity may contribute to inflammation and cell death, rendering plaque vulnerable to rupture. METHODS AND RESULTS: This study compared the effects of 12 months of treatment with darapladib (an oral Lp-PLA(2) inhibitor, 160 mg daily) or placebo on coronary atheroma deformability (intravascular ultrasound palpography) and plasma high-sensitivity C-reactive protein in 330 patients with angiographically documented coronary disease. Secondary end points included changes in necrotic core size (intravascular ultrasound radiofrequency), atheroma size (intravascular ultrasound gray scale), and blood biomarkers. BACKGROUND: =0.37). In contrast, Lp-PLA(2) activity was inhibited by 59% with darapladib (P<0.001 versus placebo). After 12 months, there were no significant differences between groups in plaque deformability (P=0.22) or plasma high-sensitivity C-reactive protein (P=0.35). In the placebo-treated group, however, necrotic core volume increased significantly (4.5+/-17.9 mm(3); P=0.009), whereas darapladib halted this increase (-0.5+/-13.9 mm(3); P=0.71), resulting in a significant treatment difference of -5.2 mm(3) (P=0.012). These intraplaque compositional changes occurred without a significant treatment difference in total atheroma volume (P=0.95). CONCLUSIONS: Despite adherence to a high level of standard-of-care treatment, the necrotic core continued to expand among patients receiving placebo. In contrast, Lp-PLA(2) inhibition with darapladib prevented necrotic core expansion, a key determinant of plaque vulnerability. These findings suggest that Lp-PLA(2) inhibition may represent a novel therapeutic approach.

Real-time photoacoustic assessment of radiofrequency ablation lesion formation in the left atrium.

In interventional electrophysiology, catheter-based radiofrequency (RF) ablation procedures restore cardiac heart rhythm by interrupting aberrant conduction paths. Real-time feedback on lesion formation and post-treatment lesion assessment could overcome procedural challenges related to ablation of underlying structures and lesion gaps. This study aims to evaluate real-time visualization of lesion progression and continuity during intra-atrial ablation with photoacoustic (PA) imaging, using clinically deployable technology. A PA-enabled RF ablation catheter was used to ablate and illuminate porcine left atrium, both excised and intact in a passive beating heart ex-vivo, for photoacoustic signal generation. PA signals were received with an intracardiac echography catheter. Using the ratio of PA images acquired with excitation wavelengths of 790 nm and 930 nm, ablation lesions were successfully imaged through circulating saline and/or blood, and lesion gaps were identified in real-time. PA-based assessment of RF-ablation lesions was successful in a realistic preclinical model of atrial intervention.

In vivo intravascular photoacoustic imaging of plaque lipid in coronary atherosclerosis.

Prospective identification of lipid-rich vulnerable plaque has remained an elusive goal. Intravascular photoacoustics, a hybrid optical and ultrasonic technology, was developed as a tool for lipid-rich plaque imaging. Here, we present the first in vivo images of lipid-rich coronary atherosclerosis acquired with this new technology in a large animal model, and relate them to independent catheter-based imaging and histology.

Functional Ultrasound (fUS) During Awake Brain Surgery: The Clinical Potential of Intra-Operative Functional and Vascular Brain Mapping.

BACKGROUND AND PURPOSE: Oncological neurosurgery relies heavily on making continuous, intra-operative tumor-brain delineations based on image-guidance. Limitations of currently available imaging techniques call for the development of real-time image-guided resection tools, which allow for reliable functional and anatomical information in an intra-operative setting. Functional ultrasound (fUS), is a new mobile neuro-imaging tool with unprecedented spatiotemporal resolution, which allows for the detection of small changes in blood dynamics that reflect changes in metabolic activity of activated neurons through neurovascular coupling. We have applied fUS during conventional awake brain surgery to determine its clinical potential for both intra-operative functional and vascular brain mapping, with the ultimate aim of achieving maximum safe tumor resection. METHODS: During awake brain surgery, fUS was used to image tumor vasculature and task-evoked brain activation with electrocortical stimulation mapping (ESM) as a gold standard. For functional imaging, patients were presented with motor, language or visual tasks, while the probe was placed over (ESM-defined) functional brain areas. For tumor vascular imaging, tumor tissue (pre-resection) and tumor resection cavity (post-resection) were imaged by moving the hand-held probe along a continuous trajectory over the regions of interest. RESULTS: A total of 10 patients were included, with predominantly intra-parenchymal frontal and temporal lobe tumors of both low and higher histopathological grades. fUS was able to detect (ESM-defined) functional areas deep inside the brain for a range of functional tasks including language processing. Brain tissue could be imaged at a spatial and temporal resolution of 300 µm and 1.5-2.0 ms respectively, revealing real-time tumor-specific, and healthy vascular characteristics. CONCLUSION: The current study presents the potential of applying fUS during awake brain surgery. We illustrate the relevance of fUS for awake brain surgery based on its ability to capture both task-evoked functional cortical responses as well as differences in vascular characteristics between tumor and healthy tissue. As current neurosurgical practice is still pre-dominantly leaning on inherently limited pre-operative imaging techniques for tumor resection-guidance, fUS enters the scene as a promising alternative that is both anatomically and physiologically informative.

Combined Confocal Microscope and Brandaris 128 Ultra-High-Speed Camera.

Controlling microbubble-mediated drug delivery requires the underlying biological and physical mechanisms to be unraveled. To image both microbubble oscillation upon ultrasound insonification and the resulting cellular response, we developed an optical imaging system that can achieve the necessary nanosecond temporal and nanometer spatial resolutions. We coupled the Brandaris 128 ultra-high-speed camera (up to 25 million frames per second) to a custom-built Nikon A1R+ confocal microscope. The unique capabilities of this combined system are demonstrated with three experiments showing microbubble oscillation leading to either endothelial drug delivery, bacterial biofilm disruption, or structural changes in the microbubble coating. In conclusion, using this state-of-the-art optical imaging system, microbubble-mediated drug delivery can be studied with high temporal resolution to resolve microbubble oscillation and high spatial resolution and detector sensitivity to discern cellular response. Combining these two imaging technologies will substantially advance our knowledge on microbubble behavior and its role in drug delivery.

Accuracy in prediction of catheter rotation in IVUS with feature-based optical flow--a phantom study.

The quantitative assessment of and compensation for catheter rotation in intravascular ultrasound images presents a fundamental problem for noninvasive characterization of the mechanical properties of the coronary arteries. A method based on the scale-space optical flow algorithm with a feature-based weighting scheme is proposed to account for the aforementioned artifact. The computed vector field, describing the misalignment between two consecutive frames, allows the quantitative assessment of the amount of vessel wall tissue motion, which is directly related to the catheter rotation. Algorithm accuracy and robustness were demonstrated on two tissue-mimicking phantoms, subjected to controlled amount of angular deviation. The proposed method shows a great reliability in the prediction of catheter rotational motion up to 4 degrees.

An inverse method for imaging the local elasticity of atherosclerotic coronary plaques.

The rupture of thin-cap fibroatheroma (TCFA) plaques is a major cause of acute coronary events. A TCFA has a trombogenic soft lipid core, shielded from the blood stream by a thin, possibly inflamed, stiff cap. The majority of atherosclerotic plaques resemble a TCFA in terms of overall structural composition, but have a more complex, heterogeneous morphology. An assessment of the material distribution is vital for quantifying the plaque's mechanical stability and for determining the effect of plaque-stabilizing pharmaceutical agents. We describe a new automated inverse elasticity method, intravascular ultrasound (IVUS) modulography, which is capable of reconstructing a heterogeneous Young's modulus distribution. The elastogram (i.e., spatial strain distribution) of the plaque is the input for the method, and is measured using the clinically available technique, IVUS elastography. Our method incorporates a novel divide-and-conquer strategy, allowing the reconstruction of TCFAs as well as heterogeneous plaques with localized regions of soft, weakened tissue. The method was applied to ex vivo elastograms, which were simulated from the cross sections of postmortem human coronary plaques. To demonstrate the clinical feasibility of the method, measured elastograms from human atherosclerotic coronary arteries were analyzed. One elastogram was measured in vitro; the other, in vivo. The method approximated the true Young's modulus distribution of all simulated plaques, while the in vitro reconstruction was in agreement with histology. In conclusion, the IVUS modulography in combination with the IVUS elastography has strong potential to become an all-encompassing modality for detecting plaques, for assessing the information related to their rupture-proneness, and for imaging their heterogeneous elastic material composition.

Sparse Ultrasound Image Reconstruction From a Shape-Sensing Single-Element Forward-Looking Catheter.

OBJECTIVE: Minimally invasive procedures, such as intravascular and intracardiac interventions, may benefit from guidance with forward-looking (FL) ultrasound. In this work, we investigate FL ultrasound imaging using a single-element transducer integrated in a steerable catheter, together with an optical shape sensing (OSS) system. METHODS: We tested the feasibility of the proposed device by imaging the surface of a tissue-mimicking (TM) phantom and an ex vivo human carotid plaque. While manually steering the catheter tip, ultrasound A-lines are acquired at 60 Hz together with the catheter shape from the OSS system, resulting in a two-dimensional sparse and irregularly sampled data set. We implemented an adaptive Normalized Convolution (NC) algorithm to interpolate the sparse data set by applying an anisotropic Gaussian kernel that is rotated according to the local direction of the catheter scanning pattern. To choose the Gaussian widths tangential ( ${\sigma _t}$) and normal ( ${\sigma _n}$) to the scanning pattern, an exhaustive search was implemented based on RMSE computation on simulated data. RESULTS: Simulations showed that the sparse data set contains only 5% of the original information. The chosen widths, ${\sigma _n} = \text{250}\;\mu {\textrm{m}}$ and ${\sigma _t} = \text{100}\;\mu{\textrm{m}}$, are used to successfully reconstruct the surface of the phantom with a contrast ratio of 0.9. The same kernel is applied successfully to the carotid plaque data. CONCLUSION: The proposed approach enables FL imaging with a single ultrasound element, mounted on a steerable device. SIGNIFICANCE: This principle may find application in a variety of image-guided interventions, such as chronic total occlusion (CTO) recanalization.

Validation of Resting Diastolic Pressure Ratio Calculated by a Novel Algorithm and Its Correlation With Distal Coronary Artery Pressure to Aortic Pressure, Instantaneous Wave-Free Ratio, and Fractional Flow Reserve.

BACKGROUND: Instantaneous wave-free ratio (iFR) offers a reliable non-hyperemic assessment of coronary physiology but requires dedicated proprietary software with a fully automated algorithm. We hypothesized that dPR (diastolic pressure ratio), calculated with novel universal software, has a strong correlation with iFR, similar diagnostic accuracy relative to resting distal coronary artery pressure/aortic pressure and fractional flow reserve (FFR). METHODS AND RESULTS: The dPR study is an observational, retrospective, single-center cohort study including patients who underwent iFR or FFR. Dedicated software was used to calculate the dPR from Digital Imaging and Communications in Medicine (DICOM) pressure waveforms. The flat period on the pressure difference between sample (dP) to the time difference between the same sample points (dt) signal was used to detect automatically the period, where the resistance is low and constant, and to calculate the dPR, which is an average over 5 consecutive heartbeats. The software was validated by correlating iFR results with dPR. Software validation was done by comparing 78 iFR measurements in 44 patients who underwent iFR. Mean iFR and dPR were 0.91±0.10 and 0.92±0.10, respectively, with a significant linear correlation ( R=0.997; P<0.001). Diagnostic accuracy was tested in 100 patients who underwent FFR. Mean FFR, resting distal coronary artery pressure/aortic pressure, and dPR were 0.85±0.09, 0.94±0.05, and 0.93±0.07, respectively. There was a significant linear correlation between dPR and FFR ( R=0.77; P<0.001). Both distal coronary artery pressure/aortic pressure and dPR had good diagnostic accuracy in the identification of lesions with an FFR ≤0.80 (area under the curve, 0.84; 95% CI, 0.76-0.92 and 0.86; 95% CI, 0.78-0.93, respectively). CONCLUSIONS: dPR, calculated by a novel validated software tool, showed a strong linear correlation with iFR. dPR correlated well with FFR with a good diagnostic accuracy to identify positive FFR.

Robust intravascular optical coherence elastography by line correlations.

We present a new method for intravascular optical coherence elastography, which is robust against motion artefacts. It employs the correlation between adjacent lines, instead of subsequent frames. Pressure to deform the tissue is applied synchronously with the line scan rate of the optical coherence tomography (OCT) instrument. The viability of the method is demonstrated with a simulation study. We find that the root mean square (rms) error of the displacement estimate is 0.55 microm, and the rms error of the strain is 0.6%. It is shown that high-strain spots in the vessel wall, such as observed at the sites of vulnerable atherosclerotic lesions, can be detected with the technique.

"Compression-only" behavior of phospholipid-coated contrast bubbles.

Ultrasound contrast agents oscillate approximately linearly up to a certain pressure range where nonlinearity sets in. Nonlinear microbubble oscillations are exploited in ultrasound pulse-echo imaging as this improves the contrast-to-tissue ratio. Here we report the observation of a highly nonlinear response of phospholipid-coated contrast agents at pressures as low as 50 kPa, termed "compression-only" behavior, where the microbubbles only compress, yet hardly expand. Time-resolved bubble dynamics recorded through ultra high-speed imaging revealed that nearly 40% of the coated bubbles show "compression-only" behavior.

Local elasticity imaging of vulnerable atherosclerotic coronary plaques.

The material composition and morphology of vulnerable atherosclerotic plaque components are considered to be more important determinants of acute coronary syndromes than the degree of stenosis. Rupture of a plaque causes thrombogenic material to contact the blood, resulting in a thrombus. Rupture-prone plaques contain an inflamed thin fibrous cap covering a large soft lipid pool. Mechanically, rupture occurs when plaques cannot withstand the internal stresses induced by the pulsating blood. These stresses concentrate within/around the cap/edge, since the lipid pool cannot bear much stress. During plaque development these stresses further increase when caps become thinner, lipid pools become larger, or the difference in stiffness (modulus) between the cap and the lipid pool increases. Intravascular ultrasound (IVUS) strain elastography/palpography and IVUS modulus elastography are imaging techniques that assess local plaque elasticity (strain and modulus) based on the principle that tissue deformation (strain) by a mechanical stress is a function of its elastic properties (modulus). Combined use of these techniques provides clinicians an all-in-one modality for detecting plaques, assessing their rupture proneness and imaging their elastic material composition. This chapter describes the terminology and pathophysiology of vulnerable plaques and discusses the techniques behind, the methods for and the validations of the elasticity imaging techniques.

Real-time volumetric lipid imaging in vivo by intravascular photoacoustics at 20 frames per second.

Lipid deposition can be assessed with combined intravascular photoacoustic/ultrasound (IVPA/US) imaging. To date, the clinical translation of IVPA/US imaging has been stalled by a low imaging speed and catheter complexity. In this paper, we demonstrate imaging of lipid targets in swine coronary arteries in vivo, at a clinically useful frame rate of 20 s-1. We confirmed image contrast for atherosclerotic plaque in human samples ex vivo. The system is on a mobile platform and provides real-time data visualization during acquisition. We achieved an IVPA signal-to-noise ratio of 20 dB. These data show that clinical translation of IVPA is possible in principle.