Dual-contrast photon-counting micro-CT using iodine and a novel bismuth-based contrast agent.

Objectives.To characterize for the first timein vivoa novel bismuth-based nanoparticular contrast agent developed for preclinical applications. Then, to design and testin vivoa multi-contrast protocol for functional cardiac imaging using the new bismuth nanoparticles and a well-established iodine-based contrast agent.Approach.A micro-computed tomography scanner was assembled and equipped with a photon-counting detector. Five mice were administered with the bismuth-based contrast agent and systematically scanned over 5 h to quantify the contrast enhancement in relevant organs of interest. Subsequently, the multi-contrast agent protocol was tested on three mice. Material decomposition was performed on the acquired spectral data to quantify the concentration of bismuth and iodine in multiple structures, e.g. the myocardium and vasculature.Main results.In the vasculature, the bismuth agent provides a peak enhancement of 1100 HU and a half-life of about 260 min. After the injection, it accumulates in the liver, spleen and intestinal wall reaching a CT value of 440 HU about 5 h post injection. Phantom measurements showed that the bismuth provides more contrast enhancement than iodine for a variety of tube voltages. The multi-contrast protocol for cardiac imaging successfully allowed the simultaneous decomposition of the vasculature, the brown adipose tissue and the myocardium.Significance.The new bismuth-based contrast agent was proven to have a long circulation time suitable for preclinical applications and to provide more contrast than iodine agents. The proposed multi-contrast protocol resulted in a new tool for cardiac functional imaging. Furthermore, thanks to the contrast enhancement provided in the intestinal wall, the novel contrast agent may be used to develop further multi contrast agent protocols for abdominal and oncological imaging.

Features Found in Indocyanine Green-Based Fluorescence Optical Imaging of Inflammatory Diseases of the Hands.

Rheumatologists in Europe and the USA increasingly rely on fluorescence optical imaging (FOI, Xiralite) for the diagnosis of inflammatory diseases. Those include rheumatoid arthritis, psoriatic arthritis, and osteoarthritis, among others. Indocyanine green (ICG)-based FOI allows visualization of impaired microcirculation caused by inflammation in both hands in one examination. Thousands of patients are now documented and most literature focuses on inflammatory arthritides, which affect synovial joints and their related structures, making it a powerful tool in the diagnostic process of early undifferentiated arthritis and rheumatoid arthritis. However, it has become gradually clear that this technique has the potential to go even further than that. FOI allows visualization of other types of tissues. This means that FOI can also support the diagnostic process of vasculopathies, myositis, collagenoses, and other connective tissue diseases. This work summarizes the most prominent imaging features found in FOI examinations of inflammatory diseases, outlines the underlying anatomical structures, and introduces a nomenclature for the features and, thus, supports the idea that this tool is a useful part of the imaging repertoire in rheumatology clinical practice, particularly where other imaging methods are not easily available.

Measurement of R1 dynamics using sliding window-DESPOT.

PURPOSE: To measure longitudinal relaxation rate (R1) changes during contrast agent studies using a driven equilibrium single pulse observation of T1 (DESPOT) method with a sliding window (sw) acquisition. MATERIALS AND METHODS: A sw-DESPOT technique was implemented that uses several three-dimensional (3D) image data sets to calculate R1 with a temporal resolution of only a single data set. Different sources of systematic errors were studied in simulations, and the technique was tested in a tumor-bearing mouse using an intravascular contrast agent. RESULTS: Consistent concentration distributions of the CA were calculated with a temporal resolution of 10 s. CONCLUSION: Sw-DESPOT offers a precise and fast method to monitor the CA dynamics in 3D volumes.

Multifunctional Rare-Earth Element Nanocrystals for Cell Labeling and Multimodal Imaging.

In this work, we describe a simple solvothermal route for the synthesis of Eu3+-doped gadolinium orthovanadate nanocrystals (Eu:GdVO4-PAA) functionalized with poly(acrylic)acid (PAA), that are applicable as cell labeling probes for multimodal cellular imaging. The Eu3+ doping of the vanadate matrix provides optical functionality, due to red photoluminescence after illumination with UV light. The Gd3+ ions of the nanocrystals reduce the T1 relaxation time of surrounding water protons, allowing these nanocrystals to act as a positive MRI contrast agent with a r1 relaxivity of 1.97 mM-1 s-1. Low background levels of Eu3+, Gd3+, and V5+ in biological systems make them an excellent label for elemental microscopy by Laser Ablation (LA)-ICP-MS. Synthesis resulted in polycrystalline nanocrystals with a hydrodynamic diameter of 55 nm and a crystal size of 36.7 nm, which were further characterized by X-ray diffraction (XRD), photoluminescence spectroscopy (PL) and transmission electron microscopy (TEM). The multifunctional nanocrystals were subsequently used for intracellular labeling of both human adipose-derived stem cells (MSCs) and A549 (adenocarcinomic human alveolar basal epithelial) cells.

In Vivo Quantification of Myocardial Infarction in Mice Using Micro-CT and a Novel Blood Pool Agent.

We herein developed a micro-CT method using the innovative contrast agent ExiTron™ MyoC 8000 to longitudinally monitor cardiac processes in vivo in small animals. Experiments were performed on healthy mice and mice with myocardial infarction inflicted by ligation of the left anterior descending artery. Time-dependent signal enhancement in different tissues of healthy mice was measured and various contrast agent doses were investigated so as to determine the minimum required dose for imaging of the myocardium. Due to its ability to be taken up by healthy myocardium but not by infarct tissue, ExiTron MyoC 8000 enables detection of myocardial infarction even at a very low dose. The signal enhancement in the myocardium of infarcted mice after contrast agent injection was exploited for quantification of infarct size. The values of infarct size obtained from the imaging method were compared with those obtained from histology and showed a significant correlation (R2 = 0.98). Thus, the developed micro-CT method allows for monitoring of a variety of processes such as cardiac remodeling in longitudinal studies.

The in vitro stability of air-filled polybutylcyanoacrylate microparticles.

Different methods of manufacturing permitted the production of air-filled PBCA microparticles (af-pbca-mp) with different physical properties such as size and wall thickness. These differences led to distinctions with respect to mechanical stability and, at the same time, to different levels of biochemical stability when incubated in biofluids. Microparticles, designed as they are to be mechanically more stable (composed of larger nanoparticles resulting in thicker shell wall, no surface hydrolysis), persist longer under in vitro conditions in biofluids such as serum, plasma and whole blood than do the more fragile ones. It was possible when using the measurement of ultrasound attenuation to characterize af-pbca-mp degradation with respect to the disappearance of the ultrasound properties of the particles and therefore to find out how long different formulations can be expected to be active as contrast agents under simulated in vivo conditions. The present examination showed that using either serum, plasma or whole blood leads to results with the same tendencies in terms of the stability and durability of af-pbca-mp in the media, mimicking in vivo conditions. It was thus possible to validate successfully the use of either serum or plasma as substitutes for whole blood. Further studies dealing with the in vitro in vivo correlation will be needed to find out if the situation in this in vitro assay corresponds to the situation in the body.

Sensitive particle acoustic quantification (SPAQ): a new ultrasound-based approach for the quantification of ultrasound contrast media in high concentrations.

RATIONALE AND OBJECTIVES: Ultrasound contrast media (USCM) consisting of gas-filled microparticles (MPs) can be detected in tissue in extremely small amounts using the stimulated acoustic emission effect (SAE), which occurs after the destruction of MPs in an acoustic field. Limited by the spatial resolution of ultrasound devices, the displayed size of individual MPs/SAEs is in the range of millimeters rather than micrometers. Thus, more than approximately 1000 microparticles per milliliter led to complete SAE saturation in the image and cannot be quantified. We have developed a method to quantify microparticles in high concentrations by increasing the resolution. METHODS: We quantified gas-filled microparticles in an agar phantom containing 30,000 microparticles per mL with a defined overlap of consecutive images, thereby destroying the microparticles with high mechanical index and measuring the corresponding SAE effects using videodensitometry. RESULTS: In each image, only those particles that had not been previously destroyed were detected. The thickness of the slices containing SAE signals was thus determined by frame-to-frame displacement. Based on the reduced slice thickness and the resulting improved spatial resolution, individual microparticles were detected even in high microparticle concentrations. CONCLUSION: Sensitive particle acoustic quantification (SPAQ) allows the quantification of microparticles, even in high concentrations, based on a massive increase in resolution.

Stabilization of indocyanine green by encapsulation within micellar systems.

Indocyanine green (ICG) is a fluorescence dye that is widely used for near-infrared imaging. Application of this dye is limited by its numerous disadvantageous properties in aqueous solution, including its concentration-dependent aggregation, poor aqueous stability in vitro and low quantum yield. Additionally, ICG is highly bound to nonspecific plasma proteins, leading to rapid elimination from the body with a half-life of 3-4 min. In this study, encapsulation of ICG within various micellar systems was investigated with the aim of overcoming these limitations. The aggregation behavior of different aqueous ICG formulations was studied using cryogenic transmission electron microscopy (cryo-TEM) and absorption spectroscopy. The micellar systems were characterized by their optical properties, particle size distribution, zeta potential and hemolytic activity. Encapsulation efficiency was determined using analytical ultracentrifugation. The best results were achieved for ICG encapsulated within aqueous Solutol HS 15 micelles. This formulation exhibited a lower aggregation behavior, a 3-fold increased quantum yield and high aqueous stability (over 4 weeks) compared to free aqueous ICG. The micelles were found to have an average diameter of 12 nm and a zeta potential close to zero (-2.1 +/- 1.7 mV). Encapsulation efficiency of ICG was high at 95%. The formulation did not display significant hemolytic activity. Consequently, Solutol HS 15 micelles are suitable nanocarriers for ICG which improve the optical properties and stability of the dye.

Characterization of superparamagnetic iron oxide nanoparticles by asymmetrical flow-field-flow-fractionation.

AIMS: The further development of diagnostic and therapeutic nanomedicines in research and their translation into clinical practice require appropriate characterization methods to ensure a reproducible quality and performance. However, many methods are insufficient for a detailed analysis of the particle size. The primary aim of the present work is to evaluate the application of asymmetrical flow-field-flow-fractionation (AF4) coupled with multiangle laser and dynamic light scattering (DLS) for the characterization of superparamagnetic iron oxide (SPIO) particles. METHODS: Eight carboxydextran-coated SPIO samples with different mean particle sizes, as determined by DLS, are investigated by means of an adequate AF4 separation method. RESULTS: In this work, we show that, with increasing sample particle size, as measured by DLS, the hydrodynamic and gyration radii obtained by the AF4 method increase respectively. We demonstrate that the applied AF4 method is able to separate nanoparticles of different sizes effectively, with superior reproducibility (relative standard deviation: <3%) and high accuracy (relative standard deviation: <10%). Furthermore, important characterization parameters that will affect the in vivo performance; namely, the shape factors and polydispersity indices, of all eight samples are presented. CONCLUSION: The work describes the application of AF4/DLS/multiangle laser light scattering as a highly useful method for characterization of SPIO particles, enabling valuable information to be accessed in addition to that obtained by transmission-electron microscopy and DLS in batch mode.

In vivo molecular imaging of adhesion molecules in experimental autoimmune encephalomyelitis (EAE).

The infiltration of autoreactive T cells into the central nervous system (CNS) requires a complex molecular interplay between immune cells and the blood-brain barrier (BBB), especially involving adhesion molecules like intercellular adhesion molecule (ICAM)-1. Here we study the molecular expression at the BBB during adoptively transferred (AT) myelin basic protein (MBP)-experimental autoimmune encephalomyelitis (EAE) in vivo by sensitive particle acoustic quantification (SPAQ)-enhanced ultrasound after intravenous application of specific gas-filled MP (MP) targeted against ICAM-1 (ICAM-MP) as contrast agent. Our results reveal a clear periventricular and cerebellar upregulation of ICAM-1 expression at the disease maximum of AT-EAE. Moreover, SPAQ-enhanced ultrasound enables the sensitive quantification of ICAM-1 expression in vivo. This allows to monitor therapeutic changes as shown by suppression of ICAM-1 expression after pretreatment of rats with corticosteroids (P < 0.008). All imaging results were confirmed by parallel immunohistochemistry. In vivo magnetic resonance imaging and albumin staining of rat brains after sonification did not reveal a disturbance of the BBB, thereby proving the safety of the method. Subsequent application of specific MP did not influence follow-up measurements, a prerequisite for sequential measurements in longitudinal studies. Based on these data, quantitative molecular imaging of adhesion molecules by SPAQ-enhanced ultrasound proves to be a safe and reliable technology to monitor changes at the BBB in vivo.

Molecular targeting of lymph nodes with L-selectin ligand-specific US contrast agent: a feasibility study in mice and dogs.

PURPOSE: To evaluate the feasibility of using intravenously administered L-selectin ligand-specific polymer-stabilized air-filled microparticles (MPs) for active targeting of peripheral lymph nodes under normal conditions in animal models. MATERIALS AND METHODS: L-selectin ligand-specific MPs and two control substances (immunoglobulin M-isotype MPs and native MPs) were each administered in three conscious mice as a single intravenous bolus injection (1.4 x 10(7) MPs/kg). All mice were sacrificed 30 minutes after administration. Lymph nodes (cervical, inguinal, axillary, popliteal, mesenteric), spleen (positive control), and kidney (blood pool control) were removed and examined for MP-related stimulated acoustic emission (SAE) signals by using harmonic color Doppler ultrasonography (US) in a tank containing degassed water. A second experiment was performed in six anesthetized beagle dogs by using the same MP formulation. Each of the MP formulations was administered in two anesthetized dogs as a single intravenous bolus injection (1 x 10(7) MPs/kg). The popliteal lymph nodes, spleen (positive control), and kidney (blood pool control) were examined in vivo with US for MP-related SAE signals 30 minutes after administration. Fisher exact test for the one-side alternative was used for mouse data analysis. RESULTS: The lymph nodes of all mice (P =.05) and the popliteal lymph nodes of both dogs treated with L-selectin ligand-specific MPs showed clear MP-related SAE signals, whereas the lymph nodes of all mice and the popliteal lymph nodes of four dogs that received the control substances did not show any SAE signals. CONCLUSION: Use of an intravenously administered L-selectin ligand-specific US contrast agent is feasible for active lymph node targeting in mice and dogs.

A real-time in vitro assay for studying functional characteristics of target-specific ultrasound contrast agents.

PURPOSE: To develop an in vitro assay for studying the feasibility of specific targeting of ultrasound contrast agents (USCAs) for ultrasound diagnostics by employing the parallel plate flow chamber, which provides an environment that mimics some aspects of the in vivo conditions like shear rate and flow effects. METHODS: USCAs based on air-filled microparticles (MP) were functionalized with specific antibodies using carbodiimide coupling chemistry and characterized by fluorescence activated cell sorter (FACS). The binding experiments were done by subjecting the MP to shear stress as they interact with the target-coated surface of the flow chamber. RESULTS: A successive modification of MP with antibody and the glass surface with antigen was achieved and quantified. The binding studies showed specific attachment of targeted MP to EDB-FN (EDB domain of fibronectin) surface. The binding of MP via nonspecific interactions was minimal. The binding efficiency of antibody-loaded MP is dependent on the applied shear stress. An increase in the wall shear stress resulted in a decrease in binding efficiency. Binding efficiency was found to be correlated with the antibody density and antigen density on the interacting surfaces. CONCLUSIONS: The results indicate that the test system developed is reliable for characterizing targeted MP without any additional labeling and can be used as a functionality assay for studying the binding characteristic of USCA with respect to different parameters like density of targeting antibodies on the microparticle surface and of target protein. In addition, the microparticles can be studied in detail under different shear rates and flow conditions. Further studies concerning the in vitro-in vivo correlation will be necessary to further increase the value of this in vitro method.