Near-Infrared Fluorescence Imaging of Carotid Plaques in an Atherosclerotic Murine Model.

Successful imaging of atherosclerosis, one of the leading global causes of death, is crucial for diagnosis and intervention. Near-infrared fluorescence (NIRF) imaging has been widely adopted along with multimodal/hybrid imaging systems for plaque detection. We evaluate two macrophage-targeting fluorescent tracers for NIRF imaging (TLR4-ZW800-1C and Feraheme-Alexa Fluor 750) in an atherosclerotic murine cohort, where the left carotid artery (LCA) is ligated to cause stenosis, and the right carotid artery (RCA) is used as a control. Imaging performed on dissected tissues revealed that both tracers had high uptake in the diseased vessel compared to the control, which was readily visible even at short exposure times. In addition, ZW800-1C's renal clearance ability and Feraheme's FDA approval puts these two tracers in line with other NIRF tracers such as ICG. Continued investigation with these tracers using intravascular NIRF imaging and larger animal models is warranted for clinical translation.

Author Correction: Non-Invasive Photoacoustic Imaging of In Vivo Mice with Erythrocyte Derived Optical Nanoparticles to Detect CAD/MI.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Non-Invasive Photoacoustic Imaging of In Vivo Mice with Erythrocyte Derived Optical Nanoparticles to Detect CAD/MI.

Coronary artery disease (CAD) causes mortality and morbidity worldwide. We used near-infrared erythrocyte-derived transducers (NETs), a contrast agent, in combination with a photoacoustic imaging system to identify the locations of atherosclerotic lesions and occlusion due to myocardial-infarction (MI). NETs (≈90 nm diameter) were fabricated from hemoglobin-depleted mice erythrocyte-ghosts and doped with Indocyanine Green (ICG). Ten weeks old male C57BL/6 mice (n = 9) underwent left anterior descending (LAD) coronary artery ligation to mimic vulnerable atherosclerotic plaques and their rupture leading to MI. 150 µL of NETs (20 µM ICG,) was IV injected via tail vein 1-hour prior to photoacoustic (PA) and fluorescence in vivo imaging by exciting NETs at 800 nm and 650 nm, respectively. These results were verified with histochemical analysis. We observed ≈256-fold higher PA signal from the accumulated NETs in the coronary artery above the ligation. Fluorescence signals were detected in LAD coronary, thymus, and liver. Similar signals were observed when the chest was cut open. Atherosclerotic lesions exhibited inflammatory cells. Liver demonstrated normal portal tract, with no parenchymal necrosis, inflammation, fibrosis, or other pathologic changes, suggesting biocompatibility of NETs. Non-invasively detecting atherosclerotic plaques and stenosis using NETs may lay a groundwork for future clinical detection and improving CAD risk assessment.

Distinctive Energy Profile of Water-Soluble, Thiolate-Protected Gold Nanoparticles as Potential Molecular Marker for Vulnerable Plaque Detection with XFCT Imaging.

Purpose: X-ray CT plays a pivotal role in diagnostic imaging, radiotherapy, and its indispensable contribution to preclinical small animal imaging research. This study characterizes a distinctive energy spectrum of a novel 3-mercaptobenzoic-acid (3MBA)-protected-144-atoms gold-nanoparticles (3MBA-Au-144-NPs) after X-ray excitation and detects vulnerable atherosclerotic plaques non-invasively using this novel contrast agent in mice carotid arteries for the first time to the best of our knowledge. Methods: We designed a four-chamber heart apex model using a 3D-printer and filled with four different concentrations of 3MBA-Au-144-NPs. The X-ray system was equipped with a pencil beam collimator, which was calibrated using a 1×1 in2 large radiochromic film. The tube was operated at 320 kVp with 12.5 mA current and multiple filtration options were available for the X-ray excitation source. The resulting pencil beam had a 3.2 mm diameter. The four-chamber apex was translated and rotated relative to the stationary pencil beam. Each sample chamber was irradiated for 2-minutes and emitted fluorescent X-rays from the excited 3MBA-Au-144-NPs were collected with CdTe and Silicon Drift (SD) detectors for 15 seconds. The spectra were used for L-shell XRF peak isolation and sonogram generation of this novel 3MBA-Au-144-NPs. The distribution and concentration of 3MBA-Au-144-NPs were reconstructed with an alternative maximum likelihood expectation maximization algorithm. For in vivo detection of unstable plaques, we developed atherosclerotic mice model after feeding them 1% high cholesterol diet (HCD) for four weeks before diabetic was induced by intraperitoneal injection of streptozotocin (STZ) to accelerate the plaque progression. Two weeks after the diabetic induction, surgically left carotid artery was ligated. Two weeks after the surgical ligation, a 250 µL of 3MBA-Au-144-NPs was IV injected after 6 hours of fasting. One hour after injection, the mice were imaged non-invasively with a cone-beam micro-CT system. Results: Two distinctive L-shell energy peaks were observed at 10 KeV and 11.13 KeV for 3MBA-Au-144-NPs in the energy spectrum of the SD detector. K-shell fluorescence events vanished in the Compton scatter and characteristic background of the tungsten source due to the lead shielding for the SD and CdTe detectors. There is a space missing at 12.5 KeV. The signal intensity varied with different 3MBA-Au-144-NPs concentration of 5%, 10%, 20%, and 100%. The X-ray fluorescence (XRF) intensity showed a highly linear response (R2=0.999) with respect to different concentrations of 3MBA-Au-144-NPs. High XRF signal was detected in the left carotid artery at 2 mm below the ligation and in aortic arch. Non-ligated right carotid artery (negative control) showed no such signal. Conclusion: These distinct energy spectra in the L-shell fluorescent energies render 3MBA-Au-144-NPs as a viable contrast agent for future in vivo XFCT imaging.

In Vivo Translation of the CIRPI System: Revealing Molecular Pathology of Rabbit Aortic Atherosclerotic Plaques.

Thin-cap fibroatheroma (TCFA) are the unstable lesions in coronary artery disease that are prone to rupture, resulting in substantial morbidity and mortality worldwide. However, their small size and complex morphologic and biologic features make early detection and risk assessment difficult. We tested our newly developed catheter-based Circumferential-Intravascular-Radioluminescence-Photoacoustic-Imaging (CIRPI) system in vivo to enable detection and characterization of vulnerable plaque structure and biology in rabbit abdominal aorta. Methods: The CIRPI system includes a novel optical probe combining circumferential radioluminescence imaging and photoacoustic tomography (PAT). The probe's CaF2:Eu-based scintillating imaging window captures radioluminescence images (360° view) of plaques by detecting ß-particles during 18F-FDG decay. A tunable laser-based PAT characterizes tissue constituents of plaque at 7 different wavelengths-540 and 560 nm (calcification), 920 nm (cholesteryl ester), 1040 nm (phospholipids), 1180 nm (elastin/collagen), 1210 nm (cholesterol), and 1235 nm (triglyceride). A single B-scan is concatenated from 330 A-lines captured during a 360° rotation. The abdominal aorta was imaged in vivo in both atherosclerotic rabbits (Watanabe Heritable Hyper Lipidemic [WHHL], 13-mo-old male, n = 5) and controls (New Zealand White, n = 2). Rabbits were fasted for 6 h before 5.55 × 107 Bq (1.5 mCi) of 18F-FDG were injected 1 h before the imaging procedure. Rabbits were anesthetized, and the right or left common carotid artery was surgically exposed. An 8 French catheter sheath was inserted into the common carotid artery, and a 0.035-cm (0.014-in) guidewire was advanced to the iliac artery, guided by x-ray fluoroscopy. A bare metal stent was implanted in the dorsal abdominal aorta as a landmark, followed by the 7 French imaging catheters that were advanced up to the proximal stent edge. Our CIRPI and clinical optical coherence tomography (OCT) were performed using pullback and nonocclusive flushing techniques. After imaging with the CIRPI system, the descending aorta was flushed with contrast agent, and OCT images were obtained with a pullback speed of 20 mm/s, providing images at 100 frames/s. Results were verified with histochemical analysis. Results: Our CIRPI system successfully detected the locations and characterized both stable and vulnerable aortic plaques in vivo among all WHHL rabbits. Calcification was detected from the stable plaque (540 and 560 nm), whereas TCFA exhibited phospholipids/cholesterol (1040 nm, 1210 nm). These findings were further verified with the clinical OCT system showing an area of low attenuation filled with lipids within TCFA. PAT images illustrated broken elastic fiber/collagen that could be verified with the histochemical analysis. All WHHL rabbits exhibited sparse to severe macrophages. Only 4 rabbits showed both moderate-to-severe level of calcifications and cholesterol clefts. However, all rabbits exhibited broken elastic fibers and collagen deposition. Control rabbits showed normal wall thickness with no presence of plaque tissue compositions. These findings were verified with OCT and histochemical analysis. Conclusion: Our novel multimodality hybrid system has been successfully translated to in vivo evaluation of atherosclerotic plaque structure and biology in a preclinical rabbit model. This system proposed a paradigm shift that unites molecular and pathologic imaging technologies. Therefore, the system may enhance the clinical evaluation of TCFA, as well as expand our understanding of coronary artery disease.

A Dual-Modality Hybrid Imaging System Harnesses Radioluminescence and Sound to Reveal Molecular Pathology of Atherosclerotic Plaques.

Atherosclerosis is a progressive inflammatory condition caused by an unstable lesion, called thin-cap fibro atheromata (TCFA) that underlies coronary artery disease (CAD)-one of the leading causes of death worldwide. Therefore, early clinical diagnosis and effective risk stratification is important for CAD management as well as preventing progression to catastrophic events. However, early detection could be difficult due to their small size, motion, obscuring 18F-FDG uptake by adjacent myocardium, and complex morphological/biological features. To overcome these limitations, we developed a catheter-based Circumferential-Intravascular-Radioluminescence-Photoacoustic-Imaging (CIRPI) system that can detect vulnerable plaques in coronary arteries and characterizes them with respect to pathology and biology. Our CIRPI system combined two imaging modalities: Circumferential Radioluminescence Imaging (CRI) and PhotoAcoustic Tomography (PAT) within a novel optical probe. The probe's CaF2:Eu based scintillating imaging window provides a 360° view of human (n = 7) and murine carotid (n = 10) arterial plaques by converting ß-particles into visible photons during 18F-FDG decay. A 60× and 63× higher radioluminescent signals were detected from the human and murine plaque inflammations, respectively, compared to the control. The system's photoacoustic imaging provided a comprehensive analysis of the plaque compositions and its morphologic information. These results were further verified with IVIS-200, immunohistochemical analysis, and autoradiography.

Imaging cellular pharmacokinetics of 18F-FDG and 6-NBDG uptake by inflammatory and stem cells.

OBJECTIVES: Myocardial infarction (MI) causes significant loss of cardiomyocytes, myocardial tissue damage, and impairment of myocardial function. The inability of cardiomyocytes to proliferate prevents the heart from self-regeneration. The treatment for advanced heart failure following an MI is heart transplantation despite the limited availability of the organs. Thus, stem-cell-based cardiac therapies could ultimately prevent heart failure by repairing injured myocardium that reverses cardiomyocyte loss. However, stem-cell-based therapies lack understanding of the mechanisms behind a successful therapy, including difficulty tracking stem cells to provide information on cell migration, proliferation and differentiation. In this study, we have investigated the interaction between different types of stem and inflammatory cells and cell-targeted imaging molecules, 18F-FDG and 6-NBDG, to identify uptake patterns and pharmacokinetics in vitro. METHODS: Macrophages (both M1 and M2), human induced pluripotent stem cells (hiPSCs), and human amniotic mesenchymal stem cells (hAMSCs) were incubated with either 18F-FDG or 6-NBDG. Excess radiotracer and fluorescence were removed and a 100 µm-thin CdWO4 scintillator plate was placed on top of the cells for radioluminescence microscopy imaging of 18F-FDG uptake, while no scintillator was needed for fluorescence imaging of 6-NBDG uptake. Light produced following beta decay was imaged with a highly sensitive inverted microscope (LV200, Olympus) and an Electron Multiplying Charge-Couple Device (EM-CCD) camera. Custom-written software was developed in MATLAB for image processing. RESULTS: The average cellular activity of 18F-FDG in a single cell of hAMSCs (0.670±0.028 fCi/µm2, P = 0.001) was 20% and 36% higher compared to uptake in hiPSCs (0.540±0.026 fCi/µm2, P = 0.003) and macrophages (0.430±0.023 fCi/µm2, P = 0.002), respectively. hAMSCs exhibited the slowest influx (0.210 min-1) but the fastest efflux (0.327 min-1) rate compared to the other tested cell lines for 18F-FDG. This cell line also has the highest phosphorylation but exhibited the lowest rate of de-phosphorylation. The uptake pattern for 6-NBDG was very different in these three cell lines. The average cellular activity of 6-NBDG in a single cell of macrophages (0.570±0.230 fM/µm2, P = 0.004) was 38% and 14% higher compared to hiPSCs (0.350±0.160 fM/µm2, P = 0.001) and hAMSCs (0.490±0.028 fM/µm2, P = 0.006), respectively. The influx (0.276 min-1), efflux (0.612 min-1), phosphorylation (0.269 min-1), and de-phosphorylation (0.049 min-1) rates were also highest for macrophages compared to the other two tested cell lines. CONCLUSION: hAMSCs were found to be 2-3× more sensitive to 18F-FDG molecule compared to hiPSCs/macrophages. However, macrophages exhibited the most sensitivity towards 6-NBDG. Based on this result, hAMSCs targeted with 18F-FDG could be more suitable for understanding the mechanisms behind successful therapy for treating MI patients by gathering information on cell migration, proliferation and differentiation.

Scintillating balloon-enabled fiber-optic system for radionuclide imaging of atherosclerotic plaques.

UNLABELLED: Atherosclerosis underlies coronary artery disease, the leading cause of death in the United States and worldwide. Detection of coronary plaque inflammation remains challenging. In this study, we developed a scintillating balloon-enabled fiber-optic radionuclide imaging (SBRI) system to improve the sensitivity and resolution of plaque imaging using (18)F-FDG, a marker of vascular inflammation, and tested it in a murine model. METHODS: The fiber-optic system uses a Complementary Metal-Oxide Silicon (CMOS) camera with a distal ferrule terminated with a wide-angle lens. The novelty of this system is a scintillating balloon in the front of the wide-angle lens to image light from the decay of (18)F-FDG emission signal. To identify the optimal scintillating materials with respect to resolution, we calculated the modulation transfer function of yttrium-aluminum-garnet doped with cerium, anthracene, and calcium fluoride doped with europium (CaF2:Eu) phosphors using an edge pattern and a thin-line optical phantom. The scintillating balloon was then fabricated from 10 mL of silicone RTV catalyst mixed with 1 mL of base and 50 mg of CaF2:Eu per mL. The addition of a lutetium oxyorthosilicate scintillating crystal (500 µm thick) to the balloon was also investigated. The SBRI system was tested in a murine atherosclerosis model: carotid-ligated mice (n = 5) were injected with (18)F-FDG, followed by ex vivo imaging of the macrophage-rich carotid plaques and nonligated controls. Confirmatory imaging of carotid plaques and controls was also performed by an external optical imaging system and autoradiography. RESULTS: Analyses of the different phosphors showed that CaF2:Eu enabled the best resolution of 1.2 µm. The SBRI system detected almost a 4-fold-higher radioluminescence signal from the ligated left carotid artery than the nonligated right carotid: 1.63 × 10(2) ± 4.01 × 10(1) vs. 4.21 × 10(1) ± 2.09 × 10(0) (photon counts), P = 0.006. We found no significant benefit to adding a scintillating crystal to the balloon: 1.65 × 10(2) ± 4.07 × 10(1) vs. 4.44 × 10(1) ± 2.17 × 10(0) (photon counts), P = 0.005. Both external optical imaging and autoradiography confirmed the high signal from the (18)F-FDG in carotid plaques versus controls. CONCLUSION: This SBRI system provides high-resolution and sensitive detection of (18)F-FDG uptake by murine atherosclerotic plaques.

Fiber-optic system for dual-modality imaging of glucose probes 18F-FDG and 6-NBDG in atherosclerotic plaques.

BACKGROUND: Atherosclerosis is a progressive inflammatory condition that underlies coronary artery disease (CAD)-the leading cause of death in the United States. Thus, the ultimate goal of this research is to advance our understanding of human CAD by improving the characterization of metabolically active vulnerable plaques within the coronary arteries using a novel catheter-based imaging system. The aims of this study include (1) developing a novel fiber-optic imaging system with a scintillator to detect both 18F and fluorescent glucose probes, and (2) validating the system on ex vivo murine plaques. METHODS: A novel design implements a flexible fiber-optic catheter consisting of both a radio-luminescence and a fluorescence imaging system to detect radionuclide 18F-fluorodeoxyglucose (18F-FDG) and the fluorescent analog 6-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-Deoxyglucose (6-NBDG), respectively. Murine macrophage-rich atherosclerotic carotid plaques were imaged ex vivo after intravenous delivery of 18F-FDG or 6-NBDG. Confirmatory optical imaging by IVIS-200 and autoradiography were also performed. RESULTS: Our fiber-optic imaging system successfully visualized both 18F-FDG and 6-NBDG probes in atherosclerotic plaques. For 18F-FDG, the ligated left carotid arteries (LCs) exhibited 4.9-fold higher radioluminescence signal intensity compared to the non-ligated right carotid arteries (RCs) (2.6 × 10(4) ± 1.4 × 10(3) vs. 5.4 × 10(3) ± 1.3 × 10(3) A.U., P = 0.008). Similarly, for 6-NBDG, the ligated LCs emitted 4.3-fold brighter fluorescent signals than the control RCs (1.6 × 10(2) ± 2.7 × 10(1) vs. 3.8 × 10(1) ± 5.9 A.U., P = 0.002). The higher uptake of both 18F-FDG and 6-NBDG in ligated LCs were confirmed with the IVIS-200 system. Autoradiography further verified the higher uptake of 18F-FDG by the LCs. CONCLUSIONS: This novel fiber-optic imaging system was sensitive to both radionuclide and fluorescent glucose probes taken up by murine atherosclerotic plaques. In addition, 6-NBDG is a promising novel fluorescent probe for detecting macrophage-rich atherosclerotic plaques.

Micro-patterned drug delivery device for light-activated drug release.

BACKGROUND: The primary goal of this study was the fabrication, long-term stability, and measured release of a marker dye from a micro-patterned drug delivery device using (i) mechanical puncture and (ii) photodisruption with an ophthalmic Nd:YAG laser. MATERIALS AND METHODS: A drug delivery device was made from a transparent bio-compatible polymer. The device consisted of two 2.6 mm diameter reservoirs containing 10% Na fluorescein dye. The device was implanted in the rabbit's eye (n = 2) with the cap of the device facing toward the exterior of the eye. Once the animals recovered from the implant surgery, 100% anhydrous glycerol was topically applied to the eye at the implantation site to decrease light scattering in the conjunctiva and sclera. The dye was released from one of the reservoir either using a 28 G ½ needle or an ophthalmic Q-switched Nd:YAG laser. A fluorescence spectrophotometer (FS) with fiber optic probe was used to measure the half-life of the dye in the eye. Measurements of fluorescence intensity were collected until the measurements return to baseline and histology was done on the tissue surrounded the device. RESULTS: None of the devices leaked of 10% Na fluorescein dye after implant. The ablation threshold of the drug delivery device was between 6 and 10 mJ to create 100-500 µm holes. The half-life measurement of the dye was found to be 13 days at the vitreous chamber after measuring the fluorescence intensity through the dilated cornea. Histology study showed minimal immune and foreign body response such as mild inflammation. CONCLUSION: This study established that the drug delivery device seemed to elicit minimal inflammatory response and retained its fluidic content until it was released with relatively longer retention time (half-life). Thus, similar device could be used for controlled release of drugs for certain ocular diseases.

Changes in morphology and optical properties of sclera and choroidal layers due to hyperosmotic agent.

Light scattering in the normally white sclera prevents diagnostic imaging or delivery of a focused laser beam to a target in the underlying choroid layer. In this study, we examine optical clearing of the sclera and changes in blood flow resulting from the application of glycerol to the sclera of rabbits. Recovery dynamics are monitored after the application of saline. The speed of clearing for injection delivery is compared to the direct application of glycerol through an incision in the conjunctiva. Although, the same volume of glycerol was applied, the sclera cleared much faster (5 to 10 s) with the topical application of glycerol compared to the injection method (3 min). In addition, the direct topical application of glycerol spreads over a larger area in the sclera than the latter method. A diffuse optical spectroscopy system provided spectral analysis of the remitted light every two minutes during clearing and rehydration. Comparison of measurements to those obtained from phantoms with various absorption and scattering properties provided estimates of the absorption coefficient and reduced scattering coefficient of rabbit eye tissue.

Variation of fluorescence in tissue with temperature.

BACKGROUND AND OBJECTIVE: Previous studies demonstrated a decrease in fluorescence intensity as tissue temperature increased. In vitro samples were increased from room temperature and in vivo canine liver from body temperature. This study investigated variations in fluorescence intensity with temperatures starting at 14°C and compared in vivo and in vitro results for consistency. STUDY DESIGN/MATERIAL AND METHODS: A fiber optic-based noninvasive system was used to characterize the temperature effect on tissue fluorescence in hamster dorsal skin in vivo, and in sclera and cornea of enucleated pig eyes in vitro. As tissue was allowed to progress through the temperature range of 14-42°C, the spectra of auto-fluorescence with respect to temperature was sampled every 1-2 minutes. A pulsed nitrogen laser was used to excite fluorescence through a fiber optic probe with a source-detector aperture separation of 370 µm. RESULTS: Fluorescence intensity decreased as temperature increased from 14 to 42°C in a phantom containing Rhodamine B dye. Results from both in vivo and in vitro tissue followed the same trend of decreasing intensity as tissue temperature increased from 14°C. Spectral intensity lineshape changed around 450 nm due to absorption from tissue. CONCLUSION: Cooling a tissue increased fluorescence intensity of skin in vivo, in all experiments. In vitro results were consistent with in vivo measurements.

Perfusion in hamster skin treated with glycerol.

BACKGROUND AND OBJECTIVE: The objective of this article is to quantify the effect of hyper-osmotic agent (glycerol) on blood velocity in hamster skin blood vessels measured with a dynamic imaging technique, laser speckle contrast imaging (LSCI). STUDY DESIGN/MATERIALS AND METHODS: In this study a dorsal skin-flap window was implanted on the hamster skin. The hyper-osmotic drug, that is, glycerol was delivered to the skin through the open dermal end of the window model. A two-dimensional map of blood flow of skin blood vessels was obtained from the speckle contrast (SC) images. RESULTS: Preliminary studies demonstrated that hyper-osmotic agents such as glycerol not only make tissue temporarily transparent, but also reduce blood flow. The blood perfusion was measured every 3 minutes for 36-66 minutes after diffusion of anhydrous glycerol. Blood flow in small capillaries was found to be reduced significantly within 3-9 minutes. Blood flow in larger blood vessels (i.e., all arteries and veins) decreased over time and some veins had significantly reduced blood flow within 36 minutes. At 24 hours, there was a further reduction in capillary blood perfusion whereas larger blood vessels regained flow compared to an hour after initial application of glycerol. CONCLUSION: Blood flow velocity and vessel diameter of the micro-vasculatures of hamster skin were reduced by the application of 100% anhydrous glycerol. At 24 hours, capillary perfusion remained depressed.

In Vivo Detection of Gold Nanoshells in Tumors Using Diffuse Optical Spectroscopy.

This study demonstrates the use of diffuse optical spectroscopy (DOS) for the noninvasive measurement of gold nanoshell concentrations in tumors of live mice. We measured the diffuse optical spectra (500-800 nm) using an optical fiber probe placed in contact with the tissue surface. We performed in vitro studies on tissue phantoms illustrating an accurate measurement of gold-silica nanoshell concentration within 12.6% of the known concentration. In vivo studies were performed on a mouse xenograft tumor model. DOS spectra were measured at preinjection, immediately postinjection, 1 and 24 h postinjection times, and the nanoshell concentrations were verified using neutron activation analysis.