Multispectral Optoacoustic Tomography of Matrix Metalloproteinase Activity in Vulnerable Human Carotid Plaques

Elevated expression of cathepsins, integrins and matrix metalloproteinases (MMPs) is typically associated with atherosclerotic plaque instability. While fluorescent tagging of such molecules has been amply demonstrated, no imaging method was so far shown capable of resolving these inflammation-associated tags with high fidelity and resolution beyond microscopic depths. This study is aimed at demonstrating a new method with high potential for noninvasive clinical cardiovascular diagnostics of vulnerable plaques using high-resolution deep-tissue multispectral optoacoustic tomography (MSOT) technology. MMP-sensitive activatable fluorescent probe (MMPSense (TM) 680) was applied to human carotid plaques from symptomatic patients. Atherosclerotic activity was detected by tuning MSOT wavelengths to activation-dependent absorption changes of the molecules, structurally modified in the presence of enzymes. MSOT analysis simultaneously provided morphology along with heterogeneous MMP activity with better than 200 micron resolution throughout the intact plaque tissue. The results corresponded well with epi-fluorescence images made from thin cryosections. Elevated MMP activity was further confirmed by zymography, accompanied by increased macrophage influx. We demonstrated, for the first time to our knowledge, the ability of MSOT to provide volumetric images of activatable molecular probe distribution deep within optically diffuse tissues. High-resolution mapping of MMP activity was achieved deep in the vulnerable plaque of intact human carotid specimens. This performance directly relates to pre-clinical screening applications in animal models and to clinical decision potential as it might eventually allow for highly specific visualization and staging of plaque vulnerability thus impacting therapeutic clinical decision making

Fast Multispectral Optoacoustic Tomography (MSOT) for Dynamic Imaging of Pharmacokinetics and Biodistribution in Multiple Organs

The characterization of pharmacokinetic and biodistribution profiles is an essential step in the development process of new candidate drugs or imaging agents. Simultaneously, the assessment of organ function related to the uptake and clearance of drugs is of great importance. To this end, we demonstrate an imaging platform capable of high-rate characterization of the dynamics of fluorescent agents in multiple organs using multispectral optoacoustic tomography (MSOT). A spatial resolution of approximately 150 µm through mouse cross-sections allowed us to image blood vessels, the kidneys, the liver and the gall bladder. In particular, MSOT was employed to characterize the removal of indocyanine green from the systemic circulation and its time-resolved uptake in the liver and gallbladder. Furthermore, it was possible to track the uptake of a carboxylate dye in separate regions of the kidneys. The results demonstrate the acquisition of agent concentration metrics at rates of 10 samples per second at a single wavelength and 17 s per multispectral sample with 10 signal averages at each of 5 wavelengths. Overall, such imaging performance introduces previously undocumented capabilities of fast, high resolution in vivo imaging of the fate of optical agents for drug discovery and basic biological research

Motion clustering for deblurring multispectral optoacoustic tomography images of the mouse heart.

Cardiac imaging in small animals is a valuable tool in basic biological research and drug discovery for cardiovascular disease. Multispectral optoacoustic tomography (MSOT) represents an emerging imaging modality capable of visualizing specific tissue chromophores at high resolution and deep in tissues in vivo by separating their spectral signatures. Whereas single-wavelength images can be acquired by multielement ultrasound detection in real-time imaging, using multiple wavelengths at separate times can lead to image blurring due to motion during acquisition. Therefore, MSOT imaging of the heart results in degraded resolution because of the heartbeat. In this work, we applied a clustering algorithm, k-means, to automatically separate a sequence of single-pulse images at multiple excitation wavelengths into clusters corresponding to different stages of the cardiac cycle. We then performed spectral unmixing on each cluster to obtain images of tissue intrinsic chromophores at different cardiac stages, showing reduced sensitivity to motion compared to signal averaging without clustering. We found that myocardium images of improved resolution and contrast can be achieved using MSOT motion clustering correction. The correction method presented could be generally applied to other MSOT imaging applications prone to motion artifacts, for example, by respiration and heartbeat

Multispectral optoacoustic tomography of myocardial infarction.

To investigate the feasibility of a high resolution optical imaging strategy for myocardial infarction.Near-infrared approaches to imaging cardiovascular disease enable visualization of disease-associated biological processes in vivo. However, even at the scale of small animals, the strong scattering of light prevents high resolution imaging after the first 1-2 mm of tissue, leading to degraded signal localization.Multispectral optoacoustic tomography (MSOT) was used to non-invasively image myocardial infarction (MI) in a murine model of coronary artery ligation at resolutions not possible with current deep-tissue optical imaging methods. Post-MI imaging was based on resolving the spectral absorption signature of a dendritic polyglycerol sulfate-based (dPGS) near-infrared imaging agent targeted to P- and L-selectin.In vivo imaging succeeded in detection of the agent in the injured myocardium after intravenous injection. The high anatomic resolution (<200 ?m) achieved by the described method allowed signals originating in the infarcted heart to be distinguished from uptake in adjacent regions. Histological analysis found dPGS signal in infarcted areas, originating from leukocytes and endothelial cells.MSOT imaging of myocardial infarction provides non-invasive visualization of optical contrast with a high spatial resolution that is not degraded by the scattering of light

Fast multispectral optoacoustic tomography (MSOT) for dynamic imaging of pharmacokinetics and biodistribution in multiple organs.

The characterization of pharmacokinetic and biodistribution profiles is an essential step in the development process of new candidate drugs or imaging agents. Simultaneously, the assessment of organ function related to the uptake and clearance of drugs is of great importance. To this end, we demonstrate an imaging platform capable of high-rate characterization of the dynamics of fluorescent agents in multiple organs using multispectral optoacoustic tomography (MSOT). A spatial resolution of approximately 150 µm through mouse cross-sections allowed us to image blood vessels, the kidneys, the liver and the gall bladder. In particular, MSOT was employed to characterize the removal of indocyanine green from the systemic circulation and its time-resolved uptake in the liver and gallbladder. Furthermore, it was possible to track the uptake of a carboxylate dye in separate regions of the kidneys. The results demonstrate the acquisition of agent concentration metrics at rates of 10 samples per second at a single wavelength and 17 s per multispectral sample with 10 signal averages at each of 5 wavelengths. Overall, such imaging performance introduces previously undocumented capabilities of fast, high resolution in vivo imaging of the fate of optical agents for drug discovery and basic biological research

Multiscale multispectral optoacoustic tomography by a stationary wavelet transform prior to unmixing.

Multispectral optoacoustic tomography (MSOT) utilizes broadband ultrasound detection for imaging biologically-relevant optical absorption features at a range of scales. Due to the multiscale and multispectral features of the technology, MSOT comes with distinct requirements in implementation and data analysis. In this work, we investigate the interplay between scale, which depends on ultrasonic detection frequency, and optical multispectral spectral analysis, two dimensions that are unique to MSOT and represent a previously unexplored challenge. We show that ultrasound frequency-dependent artifacts suppress multispectral features and complicate spectral analysis. In response, we employ a wavelet decomposition to perform spectral unmixing on a per-scale basis (or per ultrasound frequency band) and showcase imaging of fine-scale features otherwise hidden by low frequency components. We explain the proposed algorithm by means of simple simulations and demonstrate improved performance in imaging data of blood vessels in human subjects

siRNA liposome-gold nanorod vectors for multispectral optoacoustic tomography theranostics.

Therapeutic applications of gene silencing using siRNA have seen increasing interest over the past decade. The optimization of the delivery and biodistribution of siRNA using liposome-gold nanorod (AuNRs) nanoscale carriers can greatly benefit from adept imaging methods that can visualize the time-resolved delivery performance of such vectors. In this work, we describe the effect of AuNR length incorporated with liposomes and show their complexation with siRNA as a novel gene delivery vehicle. We demonstrate the application of multispectral optoacoustic tomography (MSOT) to longitudinally visualize the localisation of siRNA carrying liposome-AuNR hybrids within tumors. Combination of in vivo MSOT with ex vivo fluorescence cryo-slice imaging offers further insight into the siRNA transport and activity obtained

Translational optical imaging.

Optical imaging is experiencing significant technologic advances. Simultaneously, an array of specific optical imaging agents has brought new capabilities to biomedical research and is edging toward clinical use. We review progress in the translation of macroscopic optical imaging-including fluorescence-guided surgery and endoscopy, intravascular fluorescence imaging, diffuse fluorescence and optical tomography, and multispectral optoacoustics (photoacoustics)-for applications ranging from tumor resection and assessment of atherosclerotic plaques to dermatologic and breast examinations.Optical imaging could play a major role in the move from imaging of structure and morphology to the visualization of the individual biologic processes underlying disease and could, therefore, contribute to more accurate diagnostics and improved treatment efficacy

Optoacoustic Imaging of Human Vasculature: Feasibility by Using a Handheld Probe.

Purpose To investigate whether multispectral optoacoustic tomography (MSOT) developed for deep-tissue imaging in humans could enable the clinical assessment of major blood vessels and microvasculature. Materials and Methods The study was approved by the Institutional Review Board of the University Medical Center Groningen (CCMO-NL-43587) and registered in the Dutch National Trial Registry (NTR4125). The authors designed a real-time handheld optoacoustic scanner for human use, based on a concave 8-MHz transducer array, attaining 135° angular coverage. They applied a single-pulse-frame (SPF) sequence, which enabled motion insensitive optoacoustic imaging during handheld operation. SPF optoacoustic imaging was applied to imaging arteries and microvascular landmarks in the lower extremities of 10 healthy volunteers. The diameters selected microvessels were determined by measuring the full width at half maximum through the vessels in the MSOT images. Duplex ultrasonography was performed on the same landmarks in seven of the 10 volunteers for subjective comparison to the corresponding optoacoustic images. Results Optoacoustic imaging resolved blood vessels as small as 100 µm in diameter and within 1 cm depth. Additionally, MSOT provided images reflecting hemoglobin oxygen saturation in blood vessels, clearly identifying arteries and veins, and was able to identify pulsation in arteries during imaging. Larger blood vessels, specifically the tibialis posterior and the dorsalis pedis arteries, were also visualized with MSOT. Conclusion Handheld MSOT was found to be capable of clinical vascular imaging, providing visualization of major blood vessels and microvasculature and providing images of hemoglobin oxygen saturation and pulsation. (©) RSNA, 2016