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OBJECTIVES: This study aimed to systematically investigate whether plaque autofluorescence properties assessed with intravascular fluorescence lifetime imaging (FLIm) can provide qualitative and quantitative information about intimal composition and improve the characterization of atherosclerosis lesions. BACKGROUND: Despite advances in cardiovascular diagnostics, the analytical tools and imaging technologies currently available have limited capabilities for evaluating in situ biochemical changes associated with luminal surface features. Earlier studies of small number of samples have shown differences among the autofluorescence lifetime signature of well-defined lesions, but a systematic pixel-level evaluation of fluorescence signatures associated with various histological features is lacking and needed to better understand the origins of fluorescence contrast. METHODS: Human coronary artery segments (n=32) were analyzed with a bimodal catheter system combining multispectral FLIm with intravascular ultrasonography, compatible with in vivo coronary imaging. Various histologic components present along the luminal surface (200-μm depth) were systematically tabulated (12 sectors) from each serial histological section (n=204). Morphologic information provided by ultrasonography allowed for the accurate registration of imaging data with histology data. The relationships between histologic findings and FLIm parameters obtained from three spectral channels at each measurement location (n=33,980) were characterized. RESULTS: Our findings indicate that fluorescence lifetime from different spectral bands can be used to quantitatively predict the superficial presence of macrophage foam cells (mFCs) (ROC-AUC: 0.94) and extracellular lipid content in advanced lesions (lifetime increase in 540-nm band), detect superficial calcium (lifetime decrease in 450-nm band ROC-AUC: 0.90), and possibly detect lesions consistent with active plaque formation such as pathological intimal thickening and healed thrombus regions (lifetime increase in 390-nm band). CONCLUSIONS: Our findings indicate that autofluorescence lifetime provides valuable information for characterizing atherosclerotic lesions in coronary arteries. Specifically, FLIm can be used to identify key phenomena linked with plaque progression (e.g., peroxidized-lipid–rich mFC accumulation and recent plaque formation).

In endoscopic optical coherence tomography, a transparent protective sheath is used to protect the optics and tissue. However, the sheath causes astigmatism, which degrades transverse resolution and signal-to-noise ratio due to the cylindrical lens effect. Generally used methods for correcting this astigmatism are complex, difficult to control precisely, high-cost, and increase the dimensions of the imaging probe. To overcome these problems, we have developed an astigmatism-corrected imaging probe with an epoxy window. The astigmatism is precisely and cost-effectively adjusted controlling the curvature radius of the epoxy window, which is produced by soft lithography. Using the fiber optic fusion splicing, the fabrication process is simple. The fabricated imaging probe is almost monolithic, so its diameter is similar to that of a standard single-mode fiber. We demonstrate its astigmatism-correcting performance using focal spot analysis, imaging micro-beads and a biological sample.

Background Simplified hematoxylin and eosin (H&E) staining followed by cryo-sectioning enables rapid identification of cancerous tissue within the procedure room during Mohs micrographic surgery. Yet, a faster evaluation method is desirable as the staining protocol requires physically sectioning of the tissue after freezing, which leads to prolonged sectioning time along with the frozen artifacts that may occur in frozen sectioning. Methods We present a multichannel confocal microscopy system to rapidly evaluate cancerous tissue. Using the optical sectioning capability of the confocal microscope, optically sectioned images of the freshly excised mouse tissue were acquired and converted into images resembling H&E histology. To show details of the nuclei and structure of the tissue, we applied a newly developed rapid tissue staining method using Hoechst 33342 and Eosin-Y. Line scanning and stitching was performed to overcome the limited field of view of the confocal microscope. Unlike previous confocal systems requiring an additional mechanical device to tilt the sample and match the focus of the objective lens, we developed a focus tracking method to rapidly scan large sample area. The focus tracking provides an effective means of keeping the image of the thick tissue in focus without additional devices. We then evaluated the performance of the confocal microscope to obtain optically sectioned images in thick tissue by comparing fluorescence stained slide images. We also obtained the corresponding H&E histology image to assess the potential of the system as a diagnostic tool. Results We successfully imaged freshly excised mouse organs including stomach, tumor, and heart within a few minutes using the developed multichannel confocal microscopy and the tissue staining method. Using the pseudocolor method, colors of the acquired confocal grayscale images are converted to furthermore resemble Hematoxylin and Eosin histology. Due to the focus tracking and the line scanning, optically sectioned images were obtained over the large field of view. Comparisons with H&E histology have shown that the confocal images can acquire large details such as the ventricle as well as small details such as muscle fibers and nuclei. Conclusions This study confirms the use of confocal fluorescence microscopy technique to acquire rapid pathology results using optical sectioning, line scanning and focus tracking. We anticipate that the presented method will enable intraoperative histology and significantly reduce stress on patients undergoing surgery requiring repeated histology examinations.

Rationale: Atherosclerotic plaque is a chronic inflammatory disorder involving lipid accumulation within arterial walls. In particular, macrophages mediate plaque progression and rupture. While PPARγ agonist is known to have favorable pleiotropic effects on atherogenesis, its clinical application has been very limited due to undesirable systemic effects. We hypothesized that the specific delivery of a PPARγ agonist to inflamed plaques could reduce plaque burden and inflammation without systemic adverse effects. Methods: Herein, we newly developed a macrophage mannose receptor (MMR)-targeted biocompatible nanocarrier loaded with lobeglitazone (MMR-Lobe), which is able to specifically activate PPARγ pathways within inflamed high-risk plaques, and investigated its anti-atherogenic and anti-inflammatory effects both in in vitro and in vivo experiments. Results: MMR-Lobe had a high affinity to macrophage foam cells, and it could efficiently promote cholesterol efflux via LXRα-, ABCA1, and ABCG1 dependent pathways, and inhibit plaque protease expression. Using in vivo serial optical imaging of carotid artery, MMR-Lobe markedly reduced both plaque burden and inflammation in atherogenic mice without undesirable systemic effects. Comprehensive analysis of en face aorta by ex vivo imaging and immunostaining well corroborated the in vivo findings. Conclusion: MMR-Lobe was able to activate PPARγ pathways within high-risk plaques and effectively reduce both plaque burden and inflammation. This novel targetable PPARγ activation in macrophages could be a promising therapeutic strategy for high-risk plaques.

We propose a multipoint scanning dual-detection confocal microscopy (MS-DDCM) system for fast 3D volumetric measurements. Unlike conventional confocal microscopy, MS-DDCM can accomplish surface profiling without axial scanning. Also, to rapidly obtain 2D images, the MS-DDCM employs a multipoint scanning technique, with a digital micromirror device used to produce arrays of effective pinholes, which are then scanned. The MS-DDCM is composed of two CCDs: one collects the conjugate images and the other collects nonconjugate images. The ratio of the axial response curves, measured by the two detectors, provides a linear relationship between the height of the sample surface and the ratio of the intensity signals. Furthermore, the difference between the two images results in enhanced contrast. The normalising effect of the MS-DDCM provides accurate sample heights, even when the reflectance distribution of the surface varies. Experimental results confirmed that the MS-DDCM achieved high-speed surface profiling with improved image contrast capability.

Recent studies on endothelial dysfunction in relation to vascular diseases including atherosclerosis have highlighted the key contribution of the microenvironment of endothelial cells (ECs). By mimicking the microenvironment of early atherosclerotic lesions, here, we replicate the pathophysiological phenotype and function of ECs within microchannels. Considering the elevated deposition of fibronectin (FN) in early atherosclerotic plaques and the close correlation between the vascular stiffness and the progression of atherosclerosis, we utilized FN coated hydrogels with increased stiffness for endothelial substrates within the microchannels. As a result, we demonstrated that endothelial integrity on FN coated microchannels is likely to be undermined exhibiting a random orientation in response to the applied fluid flow, notable disruption of vascular endothelial cadherins (VE-cadherins), and higher endothelial permeability as opposed to that on microchannels coated with collagen (CL), the atheroresistant vascular model.

We integrated two-photon microscopy (TPM), second harmonic imaging microscopy (SHIM), near-infrared fluorescence microscopy (NIRF), confocal laser scanning microscopy (CLSM), and fluorescence lifetime imaging microscopy (FLIM) in one microscope system. Using our microscope system, five different images were obtained simultaneously.

Multimodal optical coherence tomography (OCT) techniques are promising diagnostic tools to accurately assess high-risk atherosclerotic plaques. For rapid translation into clinical practice, the techniques should be performed through an intravascular imaging catheter without exogenous contrast agents under the same procedures as conventional imaging. In this study, we developed a label-free, multispectral, and catheter-based imaging system to simultaneously visualize the morphological and compositional information of coronary plaques by combining fluorescence lifetime imaging (FLIm) and OCT. Using a broadband hybrid optical rotary joint and a dual-modal imaging catheter, intravascular combined FLIm-OCT imaging was safely performed in an in vivo atherosclerotic coronary artery of atherosclerotic swine models without any imaging agent. Along with detailed coronary microstructure by OCT, the multispectral FLIm could accurately visualize fluorescence lifetime signature of key biochemical components of plaque in vivo (lipid, macrophage, and fibrous tissue) when comparing the corresponding histopathological stained-sections and ex vivo FLIm microscopy images. Especially, significant differences in fluorescence lifetime distribution were noted between lipid and macrophage (p < 0.0001), which were mostly indistinguishable with standalone OCT. Also, fluorescence lifetime distributions were significantly different according to plaque types (normal, fibrous vs. lipid-rich inflamed plaque, p < 0.0001). With these statistical differences in plaque types and components, lipid distribution characterization and inflammation level estimation were provided in a pixel-by-pixel manner for the further assessment of the high-risk atherosclerotic plaque. This highly translatable imaging strategy can offer new opportunity for clinical intracoronary detection of high-risk plaques and will be a promising next-generation multimodal OCT technique.

Reflectance confocal microscopy is widely used for non-destructive optical three-dimensional (3D) imaging. In confocal microscopy, a stack of sequential two-dimensional (2D) images with respect to the axial position is typically needed to reconstruct a 3D image. As a result, in conventional confocal microscopy, acquisition speed is often limited by the rate of mechanical scanning in both the transverse and axial directions. We previously reported a high-speed parallel confocal detection method using a pinhole array for color 3D imaging without any mechanical scanners. Here, we report a high-speed color 3D imaging method based on patterned illumination employing a negative pinhole array, whose optical characteristics are the reverse of the conventional pinhole array for transmitting light. The negative pinhole array solves the inherent limitation of a conventional pinhole array, i.e., low transmittance, meaning brighter color images with abundant color information can be acquired. We also propose a 3D image processing algorithm based on the 2D cross-correlation between the acquired image and filtering masks, to produce an axial response. By using four-different filtering masks, we were able to increase the sampling points in calculation of height and enhance the lateral resolution of the color acquisition by a factor of four. The feasibility of high-speed non-contact color 3D measurement with the improved lateral resolution and brightness provided by the negative pinhole array was demonstrated by imaging various specimens. We anticipate that this high-speed color 3D measurement technology with negative pinhole array will be a useful tool in a variety of fields where rapid and accurate non-contact measurement are required, such as industrial inspection and dental scanning.

Purpose: Intravascular optical coherence tomography (IV-OCT) is a high-resolution imaging method used to visualize the microstructure of arterial walls in vivo. IV-OCT enables the clinician to clearly observe and accurately measure stent apposition and neointimal coverage of coronary stents, which are associated with side effects such as in-stent thrombosis. In this study, the authors present an algorithm for quantifying stent apposition and neointimal coverage by automatically detecting lumen contours and stent struts in IV-OCT images. Methods: The algorithm utilizes OCT intensity images and their first and second gradient images along the axial direction to detect lumen contours and stent strut candidates. These stent strut candidates are classified into true and false stent struts based on their features, using an artificial neural network with one hidden layer and ten nodes. After segmentation, either the protrusion distance (PD) or neointimal thickness (NT) for each strut is measured automatically. In randomly selected image sets covering a large variety of clinical scenarios, the results of the algorithm were compared to those of manual segmentation by IV-OCT readers. Results: Stent strut detection showed a 96.5% positive predictive value and a 92.9% true positive rate. In addition, case-by-case validation also showed comparable accuracy for most cases. High correlation coefficients (R > 0.99) were observed for PD and NT between the algorithmic and the manual results, showing little bias (0.20 and 0.46 μm, respectively) and a narrow range of limits of agreement (36 and 54 μm, respectively). In addition, the algorithm worked well in various clinical scenarios and even in cases with a low level of stent malapposition and neointimal coverage. Conclusions: The presented automatic algorithm enables robust and fast detection of lumen contours and stent struts and provides quantitative measurements of PD and NT. In addition, the algorithm was validated using various clinical cases to demonstrate its reliability. Therefore, this technique can be effectively utilized for clinical trials on stent-related side effects, including in-stent thrombosis and in-stent restenosis.

Aims Inflammation plays essential role in development of plaque disruption and coronary stent-associated complications. This study aimed to examine whether intracoronary dual-modal optical coherence tomography (OCT)-near-infrared fluorescence (NIRF) structural–molecular imaging with indocyanine green (ICG) can estimate inflammation in swine coronary artery. Methods and results After administration of clinically approved NIRF-enhancing ICG (2.0 mg/kg) or saline, rapid coronary imaging (20 mm/s pullback speed) using a fully integrated OCT-NIRF catheter was safely performed in 12 atheromatous Yucatan minipigs and in 7 drug-eluting stent (DES)-implanted Yorkshire pigs. Stronger NIRF activity was identified in OCT-proven high-risk plaque compared to normal or saline-injected controls ( P = 0.0016), which was validated on ex vivo fluorescence reflectance imaging. In vivo plaque target-to-background ratio (pTBR) was much higher in inflamed lipid-rich plaque compared to fibrous plaque ( P < 0.0001). In vivo and ex vivo peak pTBRs correlated significantly ( P < 0.0022). In vitro cellular ICG uptake and histological validations corroborated the OCT-NIRF findings in vivo . Indocyanine green colocalization with macrophages and lipids of human plaques was confirmed with autopsy atheroma specimens. Two weeks after DES deployment, OCT-NIRF imaging detected strong NIRF signals along stent struts, which was significantly higher than baseline ( P = 0.0156). Histologically, NIRF signals in peri-strut tissue co-localized well with macrophages. Conclusion The OCT-NIRF imaging with a clinical dose of ICG was feasible to accurately assess plaque inflammation and DES-related inflammation in a beating coronary artery. This highly translatable dual-modal molecular–structural imaging strategy could be relevant for clinical intracoronary estimation of high-risk plaques and DES biology.

In this study we propose an annular-beam dual-detection confocal reflectance microscopy (ADDCRM) method for high-speed three-dimensional (3D) surface measurements without longitudinal mechanical translation. ADDCRM can measure the axial position of a sample surface using the axial response curve ratio of two photodetectors, each with a different diameter pinhole. Since the height measurement range is limited by the full-width at half-maximum (FWHM) of the ratio curve, an annular beam is used to extend the depth range without reducing the lateral resolution. The experimental results demonstrate that ADDCRM achieves high-speed surface profiling with a doubled height measuring range compared to conventional Gaussian beams.

Research trends in endoscopy have been to reduce the dimension of the system for minimally invasive diagnostics and to improve spatial resolution to the microscopic level for the detailed investigation of specimens. In developing endoscopes that meet these needs, ultrathin imaging probes such as graded index lenses and fiber bundles have been widely used. And a single imaging probe is used for both illumination and detection to maintain the small diameter of the probe unit. However, this causes a fundamental problem, that is the back-reflection noise from the surface of the imaging probes. This back-reflection noise can overwhelm signals from target objects with weak contrast, which is the case for biological tissues, and degrade image contrast to such an extent that the objects remain unresolved. Here, we present an endomicroscope free from back-reflection noise generated at an ultrathin imaging probe and yet guaranteeing microscopic spatial resolution. In our method, we send illumination through single individual core fibers in the image fiber bundle, and detect signal light by the other core fibers. By blocking the back-reflection occurring only at the core used for the illumination, we remove the back-reflection noise before it reaches the detector sensor. The transmission matrix of the fiber bundle is measured and used to reconstruct a pixelation-free and high-resolution image from the raw images captured by the other fibers, which are blurred and pixelated. We demonstrated that the proposed imaging method improved 3.2 times on the signal to noise ratio produced by the conventional illumination-detection scheme.

We present multimodal intravascular optical imaging combining optical coherence tomography and fluorescence lifetime imaging. It can provide new opportunities to investigate vascular pathobiology and diagnose cardiovascular disease, by simultaneously visualizing plaque morphology and biochemical composition.

The pathophysiological progression of chronic diseases, including atherosclerosis and cancer, is closely related to compositional changes in biological tissues containing endogenous fluorophores such as collagen, elastin, and NADH, which exhibit strong autofluorescence under ultraviolet excitation. Fluorescence lifetime imaging (FLIm) provides robust detection of the compositional changes by measuring fluorescence lifetime, which is an inherent property of a fluorophore. In this paper, we present a dual-modality system combining a multispectral analog-mean-delay (AMD) FLIm and a high-speed swept-source optical coherence tomography (OCT) to simultaneously visualize the cross-sectional morphology and biochemical compositional information of a biological tissue. Experiments using standard fluorescent solutions showed that the fluorescence lifetime could be measured with a precision of less than 40 psec using the multispectral AMD-FLIm without averaging. In addition, we performed ex vivo imaging on rabbit iliac normal-looking and atherosclerotic specimens to demonstrate the feasibility of the combined FLIm-OCT system for atherosclerosis imaging. We expect that the combined FLIm-OCT will be a promising next-generation imaging technique for diagnosing atherosclerosis and cancer due to the advantages of the proposed label-free high-precision multispectral lifetime measurement.

Thin waveguides such as graded-index lenses and fiber bundles are often used as imaging probes for high-resolution endomicroscopes. However, strong back-reflection from the end surfaces of the probes makes it difficult for them to resolve weak contrast objects, especially in the reflectance-mode imaging. Here we propose a method to spatially isolate illumination pathways from detection channels, and demonstrate wide-field reflectance imaging free from back-reflection noise. In the image fiber bundle, we send illumination light through individual core fibers and detect signals from target objects through the other fibers. The transmission matrix of the fiber bundle is measured and used to reconstruct a pixelation-free image. We demonstrated that the proposed imaging method improved 3.2 times on the signal to noise ratio produced by the conventional illumination-detection scheme.

Intraplaque hemorrhages (IPH) accelerate plaque destabilization via oxidative stress, free cholesterol secretion, macrophage accumulation, and necrotic core expansion. Macrophages in these hemoglobin-exposed segments differentiate into a distinct phenotype characterized by the expression of CD163

Micro-optical coherence tomography (μOCT) is an advanced imaging technique that acquires a three-dimensional microstructure of biological samples with a high spatial resolution, up to 1 μm, by using a broadband light source and a high numerical aperture (NA) lens. As high NA produces a short depth of focus (DOF), extending the DOF is necessary to obtain a reasonable imaging depth. However, due to the complexity of optics and the limited space, it has been challenging to fabricate endoscopic μOCT, which is essential for clinical translation. Here, we report an endoscopic μOCT probe with an extended DOF by using a binary phase spatial filter. The imaging results from latex beads demonstrated that the μOCT probe achieved an axial resolution of 2.49 μm and a lateral resolution of 2.59 μm with a DOF extended by a factor of 2. The feasibility of clinical use was demonstrated by ex vivo imaging of the rabbit iliac artery.

Background— Lipid-rich inflamed coronary plaques are prone to rupture. The purpose of this study was to assess lipid-rich inflamed plaques in vivo using fully integrated high-speed optical coherence tomography (OCT)/near-infrared fluorescence (NIRF) molecular imaging with a Food and Drug Administration–approved indocyanine green (ICG). Methods and Results— An integrated high-speed intravascular OCT/NIRF imaging catheter and a dual-modal OCT/NIRF system were constructed based on a clinical OCT platform. For imaging lipid-rich inflamed plaques, the Food and Drug Administration–approved NIRF-emitting ICG (2.25 mg/kg) or saline was injected intravenously into rabbit models with experimental atheromata induced by balloon injury and 12- to 14-week high-cholesterol diets. Twenty minutes after injection, in vivo OCT/NIRF imaging of the infrarenal aorta and iliac arteries was acquired only under contrast flushing through catheter (pullback speed up to ≤20 mm/s). NIRF signals were strongly detected in the OCT-visualized atheromata of the ICG-injected rabbits. The in vivo NIRF target-to-background ratio was significantly larger in the ICG-injected rabbits than in the saline-injected controls ( P P P r =0.85) without significant bias (0.41). Cellular ICG uptake, correlative fluorescence microscopy, and histopathology also corroborated the in vivo imaging findings. Conclusions— Integrated OCT/NIRF structural/molecular imaging with a Food and Drug Administration –approved ICG accurately identified lipid-rich inflamed atheromata in coronary-sized vessels. This highly translatable dual-modal imaging approach could enhance our capabilities to detect high-risk coronary plaques.

Reflectance confocal microscopy is a widely used optical imaging technique for non-destructive three-dimensional (3D) surface measurement. In confocal microscopy, a stack of two-dimensional (2D) images along the axial position is used for 3D reconstruction. This means the speed of 3D volumetric acquisition is limited by the beam scanning and the mechanical axial scanning. To achieve fast volumetric imaging, simultaneous multiple point scanning by parallelizing the beam instead of transverse point scanning can be considered, using a pinhole array. Previously, we developed a direct-view confocal microscope with a focus tunable lens (FTL) to produce a monochrome 3D surface profile of a sample without any mechanical scanning. Here, we report a high-speed color 3D measurement method based on parallel confocal detection. The proposed method produces a color 3D image of an object by acquiring 180 2D color images with an acquisition time of 1 second. We also visualized the color information of the object by overlaying the color obtained with a color area detector and a white LED illumination on top of the 3D surface profile. In addition, we designed an improved optical system to reduce artifacts caused by internal reflections and developed a new algorithm for noise-resistant 3D measurements. The feasibility of the proposed non-contact high-speed color 3D measurement for use in industrial or biomedical fields was demonstrated by imaging the color 3D shapes of various specimens. We anticipate that this technology can be utilized in various fields, where rapid 3D surface profiles with color information are required.

We present endoscopic focal modulation microscopy (FMM) for minimally invasive imaging of deep tissue with high contrast, and then compare the results using endoscopic confocal microscopy (CM). Deep tissue imaging is achieved using a needle-like endoscopic probe based on gradient-index (GRIN) lenses. A tissue-like phantom with fluorescent micro-beads and rat kidney tubules were imaged through the endoscopic probe with FMM. FMM effectively rejected the background signals from the out-of-focus plane, thereby enhancing the image contrast and the optical sectioning ability. The combination of the GRIN endoscopic probe and FMM provides deep tissue imaging with better contrast than endoscopic CM.

Introduction: Atherosclerosis is a process of lipid accumulation and inflammation. In particular, plaque macrophages are associated with fibrous cap destabilization and rupture. We hypothesized that the specific delivery of a PPAR-γ agonist to inflamed plaques via nanoprobe (NP) targeting macrophage mannose receptors could reduce plaque burden and inflammation. Methods and Results: Macrophage mannose receptor targetable nanoprobe (MMR-NP) was fabricated by chemically conjugating thiolated glycol chitosan with mannose-PEG-maleimide, followed by incorporating PPAR-γ agonist (lobeglitazone) into this nanoscale delivery system (MMR-Lobe). For in vivo monitoring of therapeutic response, near-infrared fluorescence NP was prepared by conjugation of Cy5.5 (ext/emi 675/694 nm) with MMR-NP. In cellular uptake study, the MMR-NP showed high affinity to mannose receptors on macrophages. MMR-Lobe attenuated LPS-induced inflammatory cytokines such as TNF-α, IL-6, and MMP-9 in RAW264.7 cells. Additionally, MMR-Lobe increased expression of ABCA1, ABCG1 and LXR-α, known as PPAR-γ regulator genes involved in cholesterol efflux, in RAW264.7 cells. Using a customized high-resolution intravital fluorescence microscope, the in vivo serial imaging of carotid atheroma in apoE-/- mice injected with MMR-Lobe (7 mg/kg, twice weekly for 4 weeks) revealed a significant decrease in plaque burden and inflammation (Figure) as compared to baseline, or non-treated controls, or p.o. Lobe treated mice (p Conclusions: MMR-Lobe was able to selectively target atheroma macrophages, and effectively reduce both plaque burden and inflammation as assessed by serial intravital optical imaging. We suggest that nanoprobe-mediated PPAR-γ agonist delivery targeting plaque macrophages holds a promising theranostic approach for high-risk atheromata.

Background: Reactive oxygen species (ROS) play a central role in cigarette smoking-induced atherogenesis. The present study aims to assess the smoking-induced acute oxidative stress within vasculatures, and evaluates whether the resveratrol, a natural polyphenol antioxidant, can counteract this ROS production, using a customized, high resolution intravital optical imaging in real-time. Methods and Results: 20-week-old male C57BL/6 mice were divided into four groups according to the preceding administration of resveratrol (R) (25mg/kg via gavage, for 7 days) and exposure to cigarette smoke (CS). To in vivo assess acute oxidative stress in blood vessels, dihydroethidium, which forms a red fluorescence (ethidium, excitation/emission: 520nm/610nm) upon reaction with ROS, was injected intraperitoneally. During CS exposure, temporal changes of fluorescence signals from the mouse cremaster muscle including vasculatures were assessed by intravital optical imaging for 15 minutes. Fluorescence signals were much more pronounced in CS exposed mice than controls (p Conclusions: The intravital optical imaging was able to in vivo estimate the dynamic changes of ROS production by CS exposure. Our data demonstrated that even a brief exposure to CS increased oxidative stress in vasculatures promptly, and the resveratrol exerts protective effects against the CS-induced acute oxidative stress.

Intravascular optical coherence tomography (IV-OCT) is a high-resolution imaging method used to visualize the microstructure of arterial walls in vivo. IV-OCT enables the clinician to clearly observe and accurately measure stent apposition and neointimal coverage of coronary stents, which are associated with side effects such as in-stent thrombosis. In this study, the authors present an algorithm for quantifying stent apposition and neointimal coverage by automatically detecting lumen contours and stent struts in IV-OCT images.The algorithm utilizes OCT intensity images and their first and second gradient images along the axial direction to detect lumen contours and stent strut candidates. These stent strut candidates are classified into true and false stent struts based on their features, using an artificial neural network with one hidden layer and ten nodes. After segmentation, either the protrusion distance (PD) or neointimal thickness (NT) for each strut is measured automatically. In randomly selected image sets covering a large variety of clinical scenarios, the results of the algorithm were compared to those of manual segmentation by IV-OCT readers.Stent strut detection showed a 96.5% positive predictive value and a 92.9% true positive rate. In addition, case-by-case validation also showed comparable accuracy for most cases. High correlation coefficients (R0.99) were observed for PD and NT between the algorithmic and the manual results, showing little bias (0.20 and 0.46 μm, respectively) and a narrow range of limits of agreement (36 and 54 μm, respectively). In addition, the algorithm worked well in various clinical scenarios and even in cases with a low level of stent malapposition and neointimal coverage.The presented automatic algorithm enables robust and fast detection of lumen contours and stent struts and provides quantitative measurements of PD and NT. In addition, the algorithm was validated using various clinical cases to demonstrate its reliability. Therefore, this technique can be effectively utilized for clinical trials on stent-related side effects, including in-stent thrombosis and in-stent restenosis.

Intravascular optical coherence tomography (IV-OCT) is a high-resolution imaging method used to visualize the internal structures of walls of coronary arteries in vivo. However, accurate characterization of atherosclerotic plaques with gray-scale IV-OCT images is often limited by various intrinsic artifacts. In this study, we present an algorithm for characterizing lipid-rich plaques with a spectroscopic OCT technique based on a Gaussian center of mass (GCOM) metric. The GCOM metric, which reflects the absorbance properties of lipids, was validated using a lipid phantom. In addition, the proposed characterization method was successfully demonstrated in vivo using an atherosclerotic rabbit model and was found to have a sensitivity and specificity of 94.3% and 76.7% for lipid classification, respectively.

We propose a new method for three-dimensional (3-D) imaging without depth scanning that we refer to as the dual-detection confocal microscopy (DDCM). Compared to conventional confocal microscopy, DDCM utilizes two pinholes of different sizes. DDCM generates two axial response curves which have different stiffness according to the pinhole diameters. The two axial response curves can draw the characteristics curve of the system which shows the relationship between the axial position of the sample and the intensity ratio. Utilizing the characteristic curve, the DDCM reconstructs a 3-D surface profile with a single 2-D scanning. The height of each pixel is calculated by the intensity ratio of the pixel and the intensity ratio curve. Since the height information can be obtained directly from the characteristic curve without depth scanning, a major advantage of DDCM over the conventional confocal microscopy is a speed. The 3-D surface profiling time is dramatically reduced. Furthermore, DDCM can measure 3-D images without the influence of the sample condition since the intensity ratio is independent of the quantum yield and reflectance. We present two types of DDCM, such as a fluorescence microscopy and a reflectance microscopy. In addition, we extend the measurement range axially by varying the pupil function. Here, we demonstrate the working principle of DDCM and the feasibility of the proposed methods.

Macrophages mediate atheroma expansion and disruption, and denote high-risk arterial plaques. Therefore, they are substantially gaining importance as a diagnostic imaging target for the detection of rupture-prone plaques. Here, we developed an injectable near-infrared fluorescence (NIRF) probe by chemically conjugating thiolated glycol chitosan with cholesteryl chloroformate, NIRF dye (cyanine 5.5 or 7), and maleimide-polyethylene glycol-mannose as mannose receptor binding ligands to specifically target a subset of macrophages abundant in high-risk plaques. This probe showed high affinity to mannose receptors, low toxicity, and allowed the direct visualization of plaque macrophages in murine carotid atheroma. After the scale-up of the MMR-NIRF probe, the administration of the probe facilitated in vivo intravascular imaging of plaque inflammation in coronary-sized vessels of atheromatous rabbits using a custom-built dual-modal optical coherence tomography (OCT)-NIRF catheter-based imaging system. This novel imaging approach represents a potential imaging strategy enabling the identification of high-risk plaques in vivo and holds promise for future clinical implications.

We propose a new structure of confocal imaging system based on a direct-view confocal microscope (DVCM) with an electrically tunable lens (ETL). Since it has no mechanical moving parts to scan both the lateral (x-y) and axial (z) directions, the DVCM with an ETL allows for highspeed 3-dimensional (3-D) imaging. Axial response and signal intensity of the DVCM were analyzed theoretically according to the pinhole characteristics. The system was designed to have an isotropic spatial resolution of 20 mu m in both lateral and axial direction with a large field of view (FOV) of 10 x 10 mm. The FOV was maintained according to the various focal shifts as a result of an integrated design of an objective lens with the ETL. The developed system was calibrated to have linear focal shift over a range of 9 mm with an applied current to the ETL. The system performance of 3-D volume imaging was demonstrated using standard height specimens and a dental plaster. (C) 2016 Optical Society of America

The trade-off between spot size and depth of focus (DOF) often limits the performance of optical systems, such as optical coherence tomography and optical tweezers. Although researchers have proposed various methods to extend the DOF in free-space optics, many are difficult to implement in miniaturized optical probes due to space limitations. In this study, we present an optical probe with an extended DOF using a binary phase spatial filter (BPSF). The BPSF pattern was fabricated on the distal tip of an optical probe with a diameter of 1 mm by replica molding soft lithography, which can be easily implemented in a miniaturized optical probe due to its simple configuration. We optimized the BPSF pattern to enhance DOF, spot diameter, and light efficiency. To evaluate the fabricated endoscopic optical probe, we measured the three-dimensional point spread function of the BPSF probe and compared it with a probe without BPSF. The BPSF probe has a spot diameter of 3.56 mu m and a DOF of 199.7 mu m, while the probe without BPSF has a spot diameter of 3.69 mu m and a DOF of 73.9 mu m, representing a DOF gain of 2.7. We anticipate that this optical probe can be used in biomedical applications, including optical imaging and optical trapping techniques. (C) 2016 Optical Society of America

Advancing our understanding of human coronary artery disease requires new methods that can be used in patients for studying atherosclerotic plaque microstructure in relation to the molecular mechanisms that underlie its initiation, progression, and clinical complications, including myocardial infarction and sudden cardiac death. Here we report a dual-modality intra-arterial catheter for simultaneous microstructural and molecular imaging in vivo using a combination of optical frequency domain imaging (OFDI) and near-infrared fluorescence (NIRF) imaging. By providing simultaneous molecular information in the context of the surrounding tissue microstructure, this novel catheter could provide new opportunities for investigating coronary atherosclerosis and stent healing, and for identifying high-risk biological and structural coronary arterial plaques in vivo.

A 47-year-old man was emergently referred from another tertiary care center for percutaneous coronary intervention to the left main coronary artery (LM). Diagnostic coronary angiography (CAG) indicated a critical stenosis at the LM ostium ([Fig. 1][1]A, arrow) and haziness in the proximal to mid–

A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.

We present an aberration corrected beam scanning stimulated emission depletion (STED) microscopy. The beam scanning is essential to get images fast for practical usages in the biological applications. In this work the beam scanning method is applied to the STED microscopy. To maintain the imaging performance optimally, the aberration induced by the optic components, especially beam scanning parts, is corrected using adaptive optics. Thus, high resolution over the diffraction limit and high scanning speed are achieved without resolution and signal loss caused by the aberration.

Introduction: Intraplaque hemorrhages (IPHs) are associated with rupture-prone plaques. Within IPHs, macrophages differentiate into a specific phenotype characterized by the expression of mannose receptors. The purpose of this study was to investigate the feasibility of intravascular optical coherence tomography (OCT)-near infrared fluorescence (NIRF) structural-molecular imaging combined with a NIRF probe targeting mannose receptors to estimate IPHs in vivo. Methods and Results: IPH was induced by injecting centrifuged autologous erythrocytes through a micro-infusion catheter into rabbit aortic plaques. Normal chow diet was continued for an additional 6 weeks for macrophage differentiation. 48 hours after intravenous injection of our newly synthesized NIRF labeled nanoprobe (ex/em 750/773 nm) targeting macrophage mannose receptors at a dose of 10 mg/kg, OCT-NIRF imaging demonstrated highly enhanced NIRF signals in IPH lesions characterized by black-dotted neovessels surrounded with heterogenous OCT signals (asterisk), suggesting the association of mannose receptor positive macrophages with intraplaque hemorrhagic transformation (Figure). FM and histologic findings from the corresponding sister sections corroborated the in vivo imagings, which demonstrated the co-localization of NIRF signals with macrophage mannose receptors and hemorrhagic compositions. Conclusions: OCT-NIRF imaging with a NIRF probe targeting mannose receptors could provide a promising novel strategy to estimate IPH-associated macrophages and detect high-risk plaques.

Introduction: Plaque inflammation is associated with fibrous cap destabilization and rupture. Hypothesis: We hypothesized that the specific delivery of a novel PPARγ agonist, lobeglitazone, to inflamed plaques via nanoparticle (NP) targeting mannose receptors on macrophages could reduce inflammation in atheromata and thus, tested it using serial optical intravital molecular imaging. Methods and Results: A novel near-infrared fluorescence (NIRF) probe was fabricated by chemically conjugating thiolated glycol chitosan with mannose-PEG, and NIRF dye Cy5.5 (ext/emi 675/694 nm) for imaging. Lobeglitazone was incorporated into NIRF-NP targeting macrophage mannose receptors (MMR). In vitro, MMR-NIRF-NP was selectively uptaken by RAW264.7 cells, and NIRF signals were estimated to be 10-fold over control group (p Conclusions: Lobeglitazone-incorporated MMR-NIRF-NP was able to selectively target and image atheroma macrophages, and effectively reduce plaque inflammation as assessed by intravital optical imaging. NP-mediated lobeglitazone delivery could be a promising therapeutic strategy for high-risk plaque stabilization.

Patients with acute coronary syndrome (ACS) are prone to ischemic stroke (IS) especially during the early phase. ACS patients are more likely to have concurrent complex carotid plaques which, when destabilized, may serve as a source of distal embolism. This study investigated whether inflammatory activity in carotid artery was increased in ACS survivors compared to chronic stable angina (CSA) patients.We prospectively enrolled 74 patients with ACS or CSA (39 ACS patients versus 35 CSA patients), and fluorodeoxyglucose positron emission tomography/computed tomography ((18)F-FDG PET/CT) was performed within 1 week after diagnosis. Carotid PET signal was quantified as standardized uptake value (SUV) and target-to-background ratio (TBR, carotid SUV/jugular venous SUV).Baseline characteristics were similar between groups. TBRs and SUVs were significantly higher in the carotid arteries of ACS patients than those of CSA patients (P.001). Systemic inflammatory biomarker correlated significantly with carotid FDG uptake (high-sensitivity C-reactive protein versus average SUV: r = .361, P = .002), and the presence of cardiovascular risk factors was also related to inflammation activity. During follow-up, 3 cerebrovascular events occurred in ACS patients (including 1 early IS in a patient with severe baseline carotid inflammation), whereas none in CSA patients (P = .057).This study provided in vivo evidence that ACS survivors might experience concurrent carotid arterial inflammation. Our findings supported the role of systemic immune activation contributing to multiarterial instability in symptomatic atherosclerosis as a possible mechanistic link between ACS and IS.

The point spread function of an objective lens of a fluorescence confocal microscope was directly measured by imaging fluorescent beads. We analysed how the measurement of the point spread function was influenced by the diameter of the fluorescent beads and how the restoration technique with a deconvolution algorithm improved the measuring performance. Numerical and experimental results are presented for a typical point spread function and a zero-centred point spread function.

We describe the design and the implementation of video-rate reflection confocal scanning microscopy (CSM) using an acousto-optical deflector (AOD) for the fast horizontal scan and a galvanometer mirror (GM) for the slow vertical scan. Design parameters of the optical system are determined for optimal resolution and contrast. The OSLO simulations show that the performances of CSM are not changed with deflection angle and the wavefront errors of the system are less than 0.012λ. To evaluate the performances of designed CSM, we do a series of tests, measuring lateral and axial resolution, real time image acquisition. Due to a higher axial resolution compared with conventional microscopy, CSM can detect the surface of sub-micrometer features. We detect 138㎚ line shape pattern with a video-rate (30 frm/sec). And 10㎚ axial resolution is archived. The lateral resolution of the topographic images will be further enhanced by differential confocal microscopy (DCM) method and computational algorithms.

In light microscopy, spatial resolution is limited by diffraction effect. Confocal microscopy has improved resolutions in both lateral and axial directions, but these are still limited by diffraction effect. Confocal self-interference microscopy (CSIM) uses interference between two perpendicularly polarized beams to enhance lateral resolution. In previous research, we proposed a calcite plate with its optic-axis perpendicular to the propagation angle and one of the boundary surfaces of the plate. This type of plate is not widely used to our knowledge. In this paper, we change the calcite plate to more common one, which is commercially available. This calcite plate has its optic axis in the plane of incidence. We analyze the characteristics of this calcite plate and numerically compare the performances of CSIM in previous research and CSIM with the commercial calcite plate. Numerical results show improved performance when using the commercial calcite plate

Background: Acute coronary syndrome is frequently caused by rupture of macrophage abundant plaques with a large lipid-rich core. The present study aimed to investigate whether a fully integrated OCT/NIRF imaging combined with a clinically available near-infrared fluorescence (NIRF) enhancing ICG can detect the inflamed, lipid-rich plaques in swine coronary atheromata whose phenotype is similar to human vulnerable fibroatheroma. Methods and Results: Accelerated atherosclerosis was made by coronary balloon denudation in alloxan induced diabetic minipigs. A rapid coronary imaging (20 mm/sec pullback speed) using a fully integrated OCT/NIRF catheter was safely performed 30 minutes after I.V. injection of ICG (2.0 mg/kg) just under contrast purge. OCT clearly identified the lipid-rich plaques with fibrous cap. Simultaneously acquired, distance-calibrated NIRF imaging detected lipid-laden macrophage signals in OCT-proven plaques (figure). The in vivo plaque target-to-background ratio (pTBR) was significantly higher in ICG-injected swine compared to non-diabetic swines or saline-injected controls (pex vivo fluorescence reflectance imaging (FRI) (figure). The in vivo and ex vivo peak pTBRs correlated significantly (pIn vitro experiments, and histopathology including fluorescence microscopic imaging and immunostaining of the plaque sections corroborated the findings in vivo . Conlusions: An OCT/NIRF imaging with a clinical use of ICG accurately identified macrophage abundant, lipid-rich coronary plaques in diabetic atheromatous minipigs. This highly translatable dual-modal molecular-structural imaging could be relevant for clinical intracoronary detection of high-risk plaques.

A nonresonant, fiber-optic raster scanning endomicroscope was developed using a quarter-tubular piezoelectric (PZT) actuator. A fiber lever mechanism was utilized to enhance the small actuation range of the tubular PZT actuator and to increase its field-of-view. Finite element method simulation of the endoscopic probe was conducted for various conditions to maximize its scanning range. After fabricating the probe using a double clad fiber, we obtained two-photon fluorescence images using raster beam scanning of the fiber. The outer diameter of the probe was 3.5 mm and its rigid distal length was 30 mm including a high numerical aperture gradient index lens. These features are sufficient for input into the instrumental channel of a commercial colonoscope or gastroscope to obtain high resolution images in vivo.

A 55-year-old man with a previous history of cerebral infarct and hypertension presented to our cardiovascular center complaining of angina in November 2011. Chronic total occlusion in the proximal left anterior descending artery was identified by coronary angiography (Figure 1A). The patient subsequently underwent percutaneous coronary intervention. Wiring was done successfully with a tapered stiff wire (Conquest Pro 20; ASAHI INTECC). After multiple predilation, a single drug-eluting stent (3.0 × 18 mm) of an open-cell design and bioabsorbable polymer (Nobori; Terumo, Tokyo, Japan) was implanted using a 3.5-mm noncompliant balloon (inflation pressure, 24 atmospheric pressure; Quantum Maverick Balloon; Boston Scientific/Scimed, Maple Grove, MN) for application of final high-pressure postdilation (Figure 1B). The patient has been asymptomatic since percutaneous coronary intervention and on dual antiplatelet therapy. A 6-month scheduled angiographic follow-up demonstrated a structural misalignment at the stent implantation site with a change in focal aneurysm (Figure 1C). Digital subtraction imaging by StentBoost (Subtract; Philips Healthcare, Best, The Netherlands) was done under the suspicion of stent distortion, but the imaging was unclear to clarify the diagnosis (Figure 1D). To determine the precise configuration of the stent, we performed intravascular optical coherence tomography (OCT). Conventional 2-dimensional (2-D) OCT revealed a focal thrombus and 2 additional aneurysms …