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Background: Autofluorescence lifetime (AFL) imaging, a robust technique that enables label-free molecular investigation of biological tissues, is being introduced into the field of cardiovascular diagnostics. However, detailed AFL characteristics of coronary arteries remain elusive and there is a lack of methodology enabling such characterization. Methods: We developed multispectral fluorescence lifetime imaging microscopy (FLIM) based on analog-mean-delay. Freshly sectioned coronary arteries and atheromas, harvested from 5 swine models, were imaged using FLIM and stained to label lipids, macrophages, collagen, and smooth muscle cells. The components were quantitated from digitized histological images and compared with the corresponding FLIM. Multispectral AFL parameters derived from 2 different spectral bands (390 nm and 450 nm) were analyzed. Results: FLIM provided a wide field-of-view, high-resolution AFL imaging of frozen sections. Principal compositions of coronary arteries, such as tunica media, tunica adventitia, elastic laminas, smooth muscle cell–enriched fibrous plaque, lipid-rich core, and foamy macrophages, were well visualized in FLIM images and were found to have each different AFL spectra. In particular, proatherogenic components including lipids and foamy macrophages exhibited significantly different AFL values compared with plaque-stabilizing collagen- or smooth muscle cell–enriched tissues ( P 0.87). Conclusions: FLIM provided detailed pixel-level AFL investigation of the complex composition of coronary artery and atheroma. Our FLIM strategy enabling an automated, comprehensive visualization of multiple plaque components from unlabeled sections will be highly useful to efficiently evaluate ex vivo samples without the need for histological staining and analysis.

Optical coherence tomography (OCT) is a cross-sectional imaging method utilizing a low coherence interferometry. The lateral resolution of the OCT is limited by the numerical aperture (NA) of the imaging lens. Using a high NA lens improves the lateral resolution but reduces the depth of focus (DOF). In this study, we propose a method to improve the lateral resolution of OCT images by end-to-end training of a deep 1-D deconvolution network without use of high-resolution images.To improve the lateral resolution of the OCT, we trained the 1-D deconvolution network using lateral profiles of OCT images and the beam spot size. We used our image-guided laparoscopic surgical tool (IGLaST) to acquire OCT images of nonbiological and biological samples ex vivo. The OCT images were then blurred by applying Gaussian functions with various full width half maximums ranging from 40 to 160 µm. The network was trained using the blurred OCT images as input and the non-blurred original OCT images as output. We quantitatively evaluated the developed network in terms of similarity and signal-to-ratio (SNR), using in-vivo images of mesenteric tissue from a porcine model that was not used for training. In addition, we performed knife-edge tests and qualitative evaluation of the network to show the lateral resolution improvement of ex-vivo and in-vivo OCT images.The proposed method showed an improvement of image quality on both blurred images and non-blurred images. When the proposed deconvolution network was applied, the similarity to the non-blurred image was improved by 1.29 times, and the SNR was improved by 1.76 dB compared to the artificially blurred images, which was superior to the conventional deconvolution method. The knife-edge tests at distances at 200 to 1000 µm from the imaging probe showed an approximately 1.2 times improvement in lateral resolution. In addition, through qualitative evaluation, it was found that the image quality of both ex-vivo and in-vivo tissue images was improved with clear structure and less noise.This study showed the ability of the 1-D deconvolution network to improve the image quality of OCT images with variable lateral resolution. We were able to train the network with a small amount of data by constraining the network in 1-D. The quantitative evaluation showed better results than conventional deconvolution methods for various amount of blurring. Qualitative evaluation showed analogous results with quantitative results. This simple yet powerful image restoration method provides improved lateral resolution and suppresses background noise, making it applicable to a variety of OCT imaging applications.

High-speed and high-resolution imaging of surface profiles is critical for the investigation of various structures and mechanical dynamics of micro- and nano-scale devices. In particular, recent emergence of various nonlinear, transient and complex mechanical dynamics, such as anharmonic vibrations in mechanical resonators, has necessitated real-time surface deformation imaging with higher axial and lateral resolutions, speed, and dynamic range. However, real-time capturing of fast and complex mechanical dynamics has been challenging, and direct time-domain imaging of displacements and mechanical motions has been a missing element in studying full-field structural and dynamic behaviours. Here, by exploiting the electro-optic sampling with a frequency comb, we demonstrate a line-scan time-of-flight (TOF) camera that can simultaneously measure the TOF changes of more than 1000 spatial coordinates with hundreds megapixels/s pixel-rate and sub-nanometre axial resolution over several millimetres field-of-view. This unique combination of performances enables fast and precise imaging of both complex structures and dynamics in three-dimensional devices and mechanical resonators.

Confocal Raman microscopy is a useful tool to observe composition and constitution of label-free samples at high spatial resolution. However, accurate characterization of microstructure of tissue and its application in diagnostic imaging are challenging due to weak Raman scattering signal and complex chemical composition of tissue. We have developed a method to improve imaging speed, diffraction efficiency, and spectral resolution of confocal Raman microscopy. In addition to the novel imaging technique, the machine learning method enables confocal Raman microscopy to visualize accurate histology of tissue sections. Here, we have demonstrated the performance of the proposed method by measuring histological classification of atherosclerotic arteries and compared the histological confocal Raman images with the conventional staining method. Our new confocal Raman microscopy enables us to comprehend the structure and biochemical composition of tissue and diagnose the buildup of atherosclerotic plaques in the arterial wall without labeling.

Imaging the Eustachian tube is challenging because of its complex anatomy and limited accessibility. This study fabricated a fiber-based optical coherence tomography (OCT) catheter and investigated its potential for assessing the Eustachian tube anatomy. A customized OCT system and an imaging catheter, termed the Eustachian OCT, were developed for visualizing the Eustachian tube. Three male swine cadaver heads were used to study OCT image acquisition and for subsequent histologic correlation. The imaging catheter was introduced through the nasopharyngeal opening and reached toward the middle ear. The OCT images were acquired from the superior to the nasopharyngeal opening before and after Eustachian tube balloon dilatation. The histological anatomy of the Eustachian tube was compared with corresponding OCT images, The new, Eustachian OCT catheter was successfully inserted in the tubal lumen without damage. Cross-sectional images of the tube were successfully obtained, and the margins of the anatomical structures including cartilage, mucosa lining, and fat could be successfully delineated. After balloon dilatation, the expansion of the cross-sectional area could be identified from the OCT images. Using the OCT technique to assess the Eustachian tube anatomy was shown to be feasible, and the fabricated OCT image catheter was determined to be suitable for Eustachian tube assessment.

Rationale: Inflammation plays a pivotal role in the pathogenesis of the acute coronary syndrome. Detecting plaques with high inflammatory activity and specifically treating those lesions can be crucial to prevent life-threatening cardiovascular events. Methods: Here, we developed a macrophage mannose receptor (MMR)-targeted theranostic nanodrug (mannose-polyethylene glycol-glycol chitosan-deoxycholic acid-cyanine 7-lobeglitazone; MMR-Lobe-Cy) designed to identify inflammatory activity as well as to deliver peroxisome proliferator-activated gamma (PPARγ) agonist, lobeglitazone, specifically to high-risk plaques based on the high mannose receptor specificity. The MMR-Lobe-Cy was intravenously injected into balloon-injured atheromatous rabbits and serial in vivo optical coherence tomography (OCT)-near-infrared fluorescence (NIRF) structural-molecular imaging was performed. Results: One week after MMR-Lobe-Cy administration, the inflammatory NIRF signals in the plaques notably decreased compared to the baseline whereas the signals in saline controls even increased over time. In accordance with in vivo imaging findings, ex vivo NIRF signals on fluorescence reflectance imaging (FRI) and plaque inflammation by immunostainings significantly decreased compared to oral lobeglitazone group or saline controls. The anti-inflammatory effect of MMR-Lobe-Cy was mediated by inhibition of TLR4/NF-κB pathway. Furthermore, acute resolution of inflammation altered the inflamed plaque into a stable phenotype with less macrophages and collagen-rich matrix. Conclusion: Macrophage targeted PPARγ activator labeled with NIRF rapidly stabilized the inflamed plaques in coronary sized artery, which could be quantitatively assessed using intravascular OCT-NIRF imaging. This novel theranostic approach provides a promising theranostic strategy for high-risk coronary plaques.

In this study, we report the in vivo imaging of reactive oxygen species (ROS)-overproducing pro-inflammatory macrophages in atherosclerotic plaques using a fluorescent ROS nanosensor. We designed the nanosensor by chemically conjugating hyaluronic acid (HA, a targeting ligand for CD44 receptor), chlorin e6 [Ce6; a near-infrared fluorescent (NIRF) dye], and a thioketal (TK) linker (ROS-degradable linker). The self-assembled nanosensor emitted weak NIRF signals in normal physiological conditions, whereas it emitted strong NIRF signals under ROS-abundant conditions. The cytocompatible nanosensor showed higher intracellular internalization via receptor-mediated endocytosis, which enabled the visualization of intracellular ROS in pro-inflammatory macrophages. Moreover, we demonstrated that the nanosensor enabled the successful targeting and imaging of CD44- and ROS-overproducing pro-inflammatory macrophages in atherosclerotic plaques, as validated by confocal microscopy and immunohistological analyses. These data suggest that our ROS nanosensor is suitable for ROS imaging in pro-inflammatory macrophages in vitro and in vivo and will be a promising approach for imaging atherosclerotic tissues and evaluating the effects of antioxidants.

Micro-optical coherence tomography (µOCT) is a novel imaging approach enabling visualization of the microstructures of biological tissues at a cellular or sub-cellular level. However, it has been challenging to develop a miniaturized flexible endoscopic µOCT probe allowing helical luminal scanning. In this study, we built a flexible endoscopic µOCT probe with an outer diameter of 1.2 mm, which acquires three-dimensional images of the arterial microstructures via helical scanning with an axial and lateral resolutions of 1.83 µm and 3.38 µm in air, respectively. Furthermore, the depth of focus of the µOCT imaging probe was extended two-fold using a binary phase spatial filter. We demonstrated that the present endoscopic µOCT could image cellular level features of a rabbit artery with high-risk atheroma and a bioresorbable scaffold-implanted swine coronary artery. This highly-translatable endoscopic µOCT will be a useful tool for investigating coronary artery disease and stent biology.

Introduction and Hypothesis: Photoactivation has emerged as an innovative therapeutic strategy for coronary high-risk plaque without metallic remnants, however, limited targetable ability of photoactivator and lack of imaging guidance hinders its application to clinics. To solve these unmet needs, we developed intravascular photoactivation strategy guided by dual-modal OCT-NIRF structural-molecular imaging using a novel theranostic NIRF-emitting photoactivator. Methods and Results: Photoactivatable NIRF-emitting theranostic agent targeting scavenger receptors on macrophages was newly synthesized. The intravascular photoactivation system based on fiber type cylindrical diffuser was newly fabricated for the therapeutic light delivery to the plaques. Forty-eight hours after intravenous injection of scale-up NIRF-emitting photoactivator (equivalent Ce6 dose of 2 mg/kg), OCT-NIRF imaging was able to exactly identify the highly inflamed targets in OCT-delineated plaques of rabbit arteries. Two weeks after therapeutic light irradiation through the catheter to the NIRF enhanced macrophages, the inflammatory NIRF signals of targeted atheroma markedly decreased as compared to the controls (Figure). By comprehensive immunofluorescence analysis, photoactivation produced a prominent increase in macrophage-associated apoptotic bodies and also induced autophagy with an enhanced efferocytosis, resulting in stabilization of the atheroma. Conclusions: Intravascular photoactivation using targeted NIRF emitting photosensitizer could stabilize the inflamed plaque under the guidance of OCT-NIRF imaging. This imaging guided therapeutic innovation will be a promising theranostic strategy for the high-risk coronary plaques.

Background Photoactivation targeting macrophages has emerged as a therapeutic strategy for atherosclerosis, but limited targetable ability of photosensitizers to the lesions hinders its applications. Moreover, the molecular mechanistic insight to its phototherapeutic effects on atheroma is still lacking. Herein, we developed a macrophage targetable near-infrared fluorescence (NIRF) emitting phototheranostic agent by conjugating dextran sulfate (DS) to chlorin e6 (Ce6) and estimated its phototherapeutic feasibility in murine atheroma. Also, the phototherapeutic mechanisms of DS-Ce6 on atherosclerosis were investigated. Results The phototheranostic agent DS-Ce6 efficiently internalized into the activated macrophages and foam cells via scavenger receptor-A (SR-A) mediated endocytosis. Customized serial optical imaging-guided photoactivation of DS-Ce6 by light illumination reduced both atheroma burden and inflammation in murine models. Immuno-fluorescence and -histochemical analyses revealed that the photoactivation of DS-Ce6 produced a prominent increase in macrophage-associated apoptotic bodies 1 week after laser irradiation and induced autophagy with Mer tyrosine-protein kinase expression as early as day 1, indicative of an enhanced efferocytosis in atheroma. Conclusion Imaging-guided DS-Ce6 photoactivation was able to in vivo detect inflammatory activity in atheroma as well as to simultaneously reduce both plaque burden and inflammation by harmonic contribution of apoptosis, autophagy, and lesional efferocytosis. These results suggest that macrophage targetable phototheranostic nanoagents will be a promising theranostic strategy for high-risk atheroma. Graphical abstract

Scanning electron microscopy (SEM) is a high-resolution imaging technique with subnanometer spatial resolution that is widely used in materials science, basic science, and nanofabrication. However, conducting SEM is rather complex due to the nature of using an electron beam and the many parameters that must be adjusted to acquire high-quality images. Only trained operators can use SEM equipment properly, meaning that the use of SEM is restricted. To broaden the usability of SEM, we propose an autofocus method for a SEM system based on a dual deep learning network, which consists of an autofocusing-evaluation network (AENet) and an autofocusing-control network (ACNet). The AENet was designed to evaluate the quality of given images, with scores ranging from 0 to 9 regardless of the magnification. The ACNet can delicately control the focus of SEM online based on the AENet’s outputs for any lateral sample position and magnification. The results of these dual networks showed successful autofocus performance on three trained samples. Moreover, the robustness of the proposed method was demonstrated by autofocusing on unseen samples. We expect that our autofocusing system will not only contribute to expanding the versatility of SEM but will also be applicable to various microscopes.

Visual Abstract, Highlights • Fluorescence lifetime imaging (FLIm) allows label-free biochemical visualization of atheromas; however, it remains unknown whether FLIm can characterize high-risk plaque features in coronary arteries in a beating heart. Also, implementation of a novel analytic methodology is required for multispectral FLIm because it yields massive biochemical readouts. • This study first demonstrated a simultaneous structural and biochemical assessment of high-risk plaques in the beating swine coronary arteries using a fully integrated optical coherence tomography-FLIm and a 2.9-F low-profile dual-modal catheter. • Biochemical components of atherosclerotic plaques, including lipids, macrophages, lipids+macrophages, and fibrotic tissues, had unique fluorescence lifetime signatures that were clearly distinguishable using multispectral FLIm. • Machine learning framework was successfully integrated with multispectral FLIm and enabled an automated, quantitative imaging of multiple key components associated with plaque destabilization., Summary Coronary plaque destabilization involves alterations in microstructure and biochemical composition; however, no imaging approach allows such comprehensive characterization. Herein, the authors demonstrated a simultaneous microstructural and biochemical assessment of high-risk plaques in the coronary arteries in a beating heart using a fully integrated optical coherence tomography and fluorescence lifetime imaging (FLIm). It was found that plaque components such as lipids, macrophages, lipids+macrophages, and fibrotic tissues had unique fluorescence lifetime signatures that were distinguishable using multispectral FLIm. Because FLIm yielded massive biochemical readouts, the authors incorporated machine learning framework into FLIm, and ultimately, their approach enabled an automated, quantitative imaging of multiple key components relevant for plaque destabilization.

With the development of advanced and minimally invasive surgical techniques, and in view of the functional and cosmetic aspects, the need for rapid and accurate diagnosis during surgery is increasing. This study was conducted to develop a tissue diagnosis method using confocal microscopy after simple tissue staining that does not require freezing and slicing. At present, fluorescence staining with confocal microscopy is not generalized for real-time diagnosis during surgery. In this paper, we propose a fluorescence staining method using Hoechst 33342 and Eosin that does not require tissue freezing and slicing. The proposed method can be used as part of a rapid tissue diagnosis method that is suitable for use in the operating room, although further research is required before it can be applied in clinical practice.

Maskless lithography based on a digital micromirror device (DMD) has the advantages of high process flexibility and a low production cost. However, due to the trade-off relationship between the pixel size and exposure area, it is challenging to achieve high resolutions and high patterning speeds at the same time, which hinders the wider application of this technology in micro- and nano-fabrication processes. In addition, micromirrors in DMDs create pixelated edges that limit the pattern quality. In this paper, we propose a novel DMD maskless lithography method to improve the pattern quality during high-speed continuous patterning by means of pulse exposure and oblique scanning processes. A unique criterion, the pixel occupancy, was devised to determine the parameters related to the pulse exposure and oblique scanning optimally. We also studied how the duty cycle of the pulse exposure affects the pattern quality. As a result, we were able to increase the scanning speed up to the speed limit considering the damage threshold of the DMD and improve the pattern quality by resolving the pixelation problem. We anticipate that this method can be used in various microfabrication fields with short product life cycles or in those that require custom designs, such as the manufacturing of PCBs, MEMS devices, and micro-optics devices, among others.

Optical microscopy has been widely used in biomedical research as it provides photophysical and photochemical information of the target in subcellular spatial resolution without requiring physical contact with the specimen. To obtain a deeper understanding of biological phenomena, several efforts have been expended to combine such optical imaging modalities into a single microscope system. However, the use of multiple light sources and detectors through separated beam paths renders previous systems extremely complicated or slow for in vivo imaging. Herein, we propose a novel high-speed multimodal optical microscope system that simultaneously visualizes five different microscopic contrasts, i.e., two-photon excitation, second-harmonic generation, backscattered light, near-infrared fluorescence, and fluorescence lifetime, using a single femtosecond pulsed laser. Our proposed system can visualize five modal images with a frame rate of 3.7 fps in real-time, thereby providing complementary optical information that enhances both structural and functional contrasts. This highly photon-efficient multimodal microscope system enables various properties of biological tissues to be assessed.

We developed a high-precision multispectral fluorescence lifetime imaging microscopy (FLIM) for label-free immune-histologic imaging of atherosclerotic plaques. With images of fluorescence lifetimes and intensity ratios between different channels, we could characterize various plaque components of coronary arteries that are related to immunohistochemistry results. Correlative FLIM-immunohistochemistry validation revealed significant associations between plaque components and multispectral FLIM parameters. The machine learning algorithm, trained with co-registered FLIM-immunohistochemistry datasets, allowed automated visualization of multiple atherosclerotic components from FLIM image of an unstained section. We anticipate that the multispectral FLIM can be widely used to assess biochemical components of various biological tissues, including atherosclerotic plaques.

Intravascular optical coherence tomography-fluorescence lifetime imaging (OCT-FLIm) provides co-registered structural and biochemical information of atherosclerotic plaques in a label-free manner. For intuitive image interpretation of OCT-FLIm, herein, we present a machine learning classifier where key biochemical components (lipids, lipids+macrophages, macrophages, fibrotic, and normal) related to plaque destabilization are characterized based on the combination of multispectral FLIm parameters and convolutional OCT features. Using dataset from in vivo atheromatous swine models, the classification accuracy was >92% for each plaque component according the five-fold cross validation. This highly translatable imaging strategy will open a new avenue for clinical intracoronary assessment of high-risk plaques.

We successfully acquired a comprehensive three-dimensional imaging of swine Eustachian tubes using a catheter-based OCT. The OCT images clearly identified the structure and tissue components of the Eustachian tube, which was confirmed by the corresponding histological images. In addition, after the balloon dilation of the Eustachian tube, the OCT images clearly visualized the expanded luminal area of the Eustachian tube, quantitatively. The catheter-based OCT is expected to be applicable to the diagnosis and research of Eustachian tube disorder by providing real-time assessment.

Photoactivation is a promising theranostic tool to image and stabilize the atherosclerotic plaque by apoptosis induction in macrophages or other vascular cells; however, lack of effective drugs and mechanistic understanding hinder its clinical application for cardiovascular disease. Here, we developed the macrophage targeted photosensitizer delivery strategy and demonstrated that imaging assisted light activation reduced inflammation and burden of atherosclerotic plaques. Mechanistically, targeted photoactivation induced autophagy and increased MerTK expression in carotid atheroma as early as 1 day, and had 2-fold increase in macrophage-associated apoptotic cells, indicating efferocytosis enhancement. This multifunctional photoactivatable theranostic strategy could confer a promising tool for high-risk plaques.

Multimodal nonlinear microscopy has been widely applied in biology and medicine due to its relatively deep penetration into tissue and its label-free manner. However, current multimodal systems require the use of multiple sources and detectors, leading to bulky, complex, and expensive systems. In this Letter, we present a novel method of using a single light source and detector for nonlinear multimodal imaging of biological samples. Using a photonic crystal fiber, a pulse picker, and multimode fibers, our developed system successfully acquired multimodal images of swine coronary arteries, including two-photon excitation fluorescence, second-harmonic generation, coherent anti-Stokes Raman scattering, and backreflection. The developed system could be a valuable tool for various biomedical applications.

A multimodal nonlinear microscope using single source and detector was developed. With the microscope we successfully acquired two-photon excited fluorescence (TPEF), second-harmonic generation (SHG), coherent anti-Stokes Raman scattering (CARS) and back reflection images of biological samples.

We demonstrated a blood flow v isualization m ethod f or t he image-guided laparoscopic surgical tool (IGLaST). Using the inter A-line OCT decorrelations from a moving probe, blood flows in a phantom were successfully isolated from the background.

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Introduction: Stress-associated neurobiological activity is associated with future cardiovascular events. However, whether the linkage between brain emotional activity and complex plaque characteristics exists during the phase of acute plaque instability needs to be elucidated. Objectives: We aimed to prospectively estimate brain amygdalar activity (AmygA), arterial inflammation (AI) and macrophage hematopoiesis (HEMA) according to the presence of complex plaque characteristics in patients with acute myocardial infarction (AMI). Methods: In a total of 45 AMI patients (mean 60.0 years, 84.4% male), 18F FDG-PET/CT imaging was performed within 45 days of index episode. Total plaque burden was assessed using the SYNTAX score. The culprit plaque was considered complex, if there were two or more of the following morphologic features: thrombus, ulceration, plaque irregularity, and impaired flow (TIMI Results: Average SYNTAX score ranged by 16.3±8.4 and 12 (26.7%) patients had intermediate to high score (≥22). Complex culprit plaque and significant flow limitation were present in 34 (75.6%) and 33 (73.3%) patients, respectively. AmygA increased significantly in those who had higher SYNTAX score (≥22; target-to-background ratio [TBR]: 0.68±0.08 vs. 0.63±0.06; p=0.004), complex culprit plaques (TBR: 0.66±0.05 vs. 0.60±0.04; p Conclusions: AmygA was markedly enhanced in AMI patients with complex plaque characteristics. These findings suggest that AmygA closely associates with loco-regional disease activity at the level of coronary vasculature.

Introduction: Autophagy plays a protective role in atherosclerosis and can promote efferocytosis of apoptotic cells. Photoactivation is a promising tool for treating cardiovascular disease, and induces autophagy in macrophages-derived foam cells, however, regulation of autophagy and efferocytosis by photoactivation in atherosclerotic plaque remains unknown. Objective: The present study aims to investigate whether macrophage targeted light activation induces autophagy and enhances efferocytosis in inflamed plaques Methods and Results: Targeted photoactivatable agent showed specific binding affinity and phototoxicity to scavenger receptor-expressing macrophages. Confocal microscopy analysis over time after irradiation demonstrated the induction of apoptosis and autophagy flux in macrophage-derived foam cells. The theranostic agent localized to macrophage scavenger receptors within atherosclerotic plaques, and intravital fluorescence microscopy imaged plaque inflammation and was used to monitor photobleaching after irradiation. Serial in vivo imaging analysis demonstrated that macrophage-specific photoactivation reduced plaque burden and inflammation after 1 week (Figure A). Mechanistically, targeted light activation induced autophagy within atheroma as early as 1 day (Figure B), and increased phagocytic receptor Mer tyrosine kinase (MerTK) expression. Targeted photoactivation had 2-fold-increase in macrophage-associated apoptotic bodies at one week after laser irradiation, indicating enhanced efferocytosis (Figure C). Conclusions: As a sensitive multifunctional therapeutic and imaging strategy, macrophage targeted photoactivation with autophagy induction could confer a promising theranostic strategy for the high-risk plaques.

Background/Objectives: Vascular spotty calcification is an actively regulated biological process resulting in plaque vulnerability. We investigated the feasibility of a novel alendronate-based near-infrared fluorescence (NIRF)-emitting probe to specifically target atherosclerotic calcification in a murine model in vivo using our customized high-resolution multichannel intravital molecular imaging system (IVFM). Methods/Results: We have fabricated a calcium-binding NIRF probe by chemically coupling alendronate, a specific targeting ligand, and NIRF-emitting Cy5.5 to the ends of azide-PEG-NHS ester (Al-Cy5.5). Prepared Al-Cy5.5 has high affinity for calcium phosphate-containing bone minerals. In vitro, Al-Cy5.5 specifically binds to RANKL-induced osteogenic-macrophages as compared to macrophages (p Conclusions: Our novel calcification targeted probe, Al-Cy5.5, was able to selectively target atheroma calcification in vivo in a murine model as assessed by optical IVFM. This novel targetable strategy is expected to provide a promising theranostic basis for calcified high-risk plaques by integration with multimodal customized catheter imaging system.