This PDF file contains the front matter associated with SPIE Proceedings Volume 10471, including the Title Page, Copyright information, Table of Contents, and Conference Committee listing.

We report the design and validation of a novel ball lens-based imaging catheter based on dual-clad fiber for frequency-domain fluorescence lifetime imaging microscopy (FLIM) of atherosclerosis. The illumination and collection performance of the catheter endoscope was modeled and optimized with the ray-tracing program Zemax. A 1.55-m-long dual-clad fiber was spliced with a short length of coreless fiber, and then heated and polished to fabricate the angled ball lens. The fiber endoscope was enclosed in a torque cable and had a diameter of 2Fr. The catheter was affixed to a custom built lensless rotary joint which had high coupling efficiency (>90%) over a broad spectral range, accommodating both the UV (375 nm) excitation and the broad fluorescence emission (385 nm - 600 nm). The computer controlled rotary joint and translation stage for pullback imaging can routinely achieve rotation rates of 6000 rpm. The endoscope has two configurations depending on different illumination methods. Lateral resolution was improved more than twice by illuminating the core instead of the inner cladding, while SNR decreased due to higher attenuation of the core. Experiments conducted using a resolution target demonstrate a lateral resolution 80 μm at 1 mm lens-to-sample distance. Experiments conducted using a fluorescein phantom and a segment of ex vivo human coronary artery demonstrate the system performance for fluorescence lifetime imaging with pullback velocities of >10mm/s. This study demonstrates the novel design of a ball lens-based FLIM catheter system to record fluorescence in a continuous helical scanning method across broad-spectral emission bands.

Bovine pericardium (BP) exhibits distinct biochemical and biomechanical properties that are dominant by the structural protein collagen. The enzymatic degradation of collagen molecules is critical for in vivo incorporation and remodeling of BP in tissue engineering applications. A non-destructive method for monitoring BP during degradation would provide a valuable tool for quantifying functional changes initially in vitro and ultimately in vivo. In this study, we demonstrated the sensitivity of multi-spectral fluorescence lifetime imaging system (ms-FLIm) developed by our group to collagen content and compressive modulus of BP during collagenase degradation. A pairwise study was performed using bacterial collagenase to partially digest BP. We measured the biomaterials properties with ms-FLIm and destructive conventional measurements including collagen assay, compressive test and histology. A single factor study design was utilized. Test group samples were digested by bacterial collagenase for 0, 8, 16 and 24 hours, while control group samples were prepared in the Hank’s balanced salt solution to control for time in solution. Statistical analysis was performed using the Kendall τB correlation test. The results demonstrate that fluorescence parameters measured by ms-FLIm are significantly correlated with collagen content and compressive modulus (|τB| > 0.45, p < 0.05). Based on these findings, we aim to predict BP’s collagen content and mechanical properties using fluorescence metrics, and ultimately apply ms-FLIm for non-destructively monitoring of in vivo remodeling of BP.

Atherosclerosis is a progressive asymptomatic disease that has the highest rate of death and morbidity in the United States. High macrophage infiltration and thin cap fibroatheromas are known to be the precursor lesions of plaque rupture. Lipid-laden macrophages called foam cells are formed by the uptake of lipids within the plaque. These foam cells eventually die forming a necrotic core. Ruptured plaques are characterized by a necrotic core with an overlying thin-ruptured cap highly infiltrated by macrophages. Imaging modalities capable of identifying macrophage clusters in atherosclerotic plaques could be used for plaque vulnerability assessment. In this study, Multispectral Fluorescence Lifetime Imaging (FLIM) is used to retrieve information of biochemical markers present in atherosclerotic tissue. Here, we present a computational methodology that makes use of FLIM-based biochemical plaque features in order to identify macrophage/foam cells in atherosclerotic plaques. In the proposed methodology, the FLIM lifetime map obtained from a spectral channel of 494 ± 20.5 nm provides information about the accumulation of macrophages, which produce long lifetimes (>6 ns). This methodology was validated against histopathological assessment (CD68 staining specific for macrophages) in terms of statistical correlation, a 10-fold cross validation (sensitivity = 88.45%; specificity= 91.21%), and receiver operating characteristic (ROC AUC = 0.91) analyses.

Techniques that dynamically assess the maturation of tissue engineered constructs allow more efficient longitudinal control of developmental parameters than traditional destructive analyses, enhancing the likelihood of successful outcomes. We present a non-destructive and minimally invasive imaging method to monitor the growth of engineered vascular tissue based on label-free fluorescence lifetime imaging (FLIm) using a single fiber optic interface. We demonstrate the potential of the fiber-based FLIm system on vascular grafts composed of antigen removed bovine pericardium extracellular scaffolds seeded with human endothelial or mesenchymal stem cells. Tissue constructs are illuminated with 355 nm pulsed laser light that excites tissue autofluorescence, stemming from scaffold proteins (e.g., collagen), and cellular metabolic co-factors (i.e., NADH and FAD). Fluorescence lifetime images are acquired by scanning the distal tip of a multimode fiber across the sample surface, to deliver fluorescence excitation and collect fluorescence emission. A wavelength selection module is used to spectrally separate autofluorescence into four spectral bands that were selected to match the emission peaks of the main tissue fluorophores. By examining the relative intensity and mean fluorescence lifetime in each spectral band we identify the composition of engineered tissues, and evaluate the progression of recellularization. The fiber-based apparatus is compatible with imaging a range of sample geometries including planar and tubular constructs, and imaging in regions of restricted space such as inside tissue bioreactors, or in vivo. Future applications for the system include longitudinal monitoring of the luminal surface of engineered vascular tissues, or intravascular imaging in vivo to monitor viability of vascular implants.

The mechanical components of the heart, especially the valves, are enormously stressed during lifetime and undergo different pathophysiological tissue transformations, which affect cardiac output and in consequence living comfort of affected patients. Calcific aortic valve stenosis (AVS) is the most common valve disease in modern industrial countries but the pathogenesis and progression of this disease is still unknown. Therefore, animal models, especially mouse models, are a powerfull tool to investigate this disease in more detail with high resolution imaging techniques like optical coherence tomography and video microscopy. A custom-made pump was used for artificial stimulation of aortic valves ex vivo of 17-week-old wildtype and 12-month-old ApoE knockout mice. Image acquisition and viszualization of tissue dynamics was perfomed by using a multimodal imaging system for time-resolved 3D OCT and high-speed microscopy. Exemplary findings will be presented showing the differences in tissue behaviour and dynamics of the aortic valves, which were visualized under same conditions of artificial stimulation with 4D OCT and high speed mi-croscopy. Furthermore, clinically relevant parameters like maximum opening area and slope time of the valve movement can be measured from these time-resolved image data. The presented results show that optical coherence tomography and high-speed video microscopy are prom-ising tools for the investigation of dynamic behavior and its changes in calcific aortic valve stenosis disease models in mice. OCT offers an easy access to the morphology in 3D and the measurement of tissue parameters like tissue thickness without any sample preparation like staining or cutting.

Acute cardiovascular events are still the leading cause of morbidity and mortality in the Western world. The rupture of a vulnerable atherosclerotic plaque is the most common cause of an acute cardiovascular event. Vulnerable plaques are characterized by presenting a necrotic core below a thin fibrous cap, and extensive infiltration of macrophages and foam cells. Thus, the degree of macrophage accumulation is an indicator in determining plaque progression and probability of rupture. This work presents a simple and fast image processing method for the identification of agglomerated macrophage/foam-cells regions in intravascular optical coherence tomography (IV-OCT) images that might be used for in-vivo assessment of plaque vulnerability. This method relies on the ratio of the values of either the normalized-intensity standard deviation or the entropy estimated over two axially adjacent regions of interest in IV-OCT images. This method is able of highlighting areas in the IV-OCT images where significant amount of macrophages is localized, and was applied to IV-OCT scans of 26 postmortem coronary segments and validated against histopathological assessment. The accuracy for detecting macrophage/foam-cell was as follows: the normalized standard deviation ratio approach showed an accuracy of 86% and a sensitivity and specificity of 85.8% and 86.1%, respectively; while the entropy ratio led to an accuracy of 86.9% and a sensitivity and specificity of 86.8% and 86.9%, respectively.

Optical coherence tomography (OCT) has been a useful clinical tool for diagnosing coronary artery disease through a flexible catheter, but its full promise relies on resolving cellular and sub-cellular structures in vivo. Previously, visualizing cellular structures through an imaging catheter is not possible due to limited depth of focus (DOF) of a tightly focused Gaussian beam: typically, a Gaussian beam with 2-3 μm resolution has a DOF within 100 μm, which is not sufficient for in vivo catheter imaging. Therefore, we developed a self-imaging wavefront division optical system that generates a coaxially-focused multimode (CAFM) beam with a DOF that is approximately one order of magnitude longer than that of a Gaussian beam. In this study, we present a high-resolution, extended DOF catheter based on self-imaging wavefront division optics. The catheter generates a CAFM beam with a lateral resolution of 3 μm and a DOF close to 2 mm. To correct the aberration introduced by catheter sheath, we incorporated a cylindrical prism to compensate the sheath astigmatism. When the catheter is incorporated into a micro-resolution OCT (μOCT) system with rotational scanning mechanics, cellular-resolution cross-sectional images of the coronary artery wall can be obtained. The device serves as an important step toward characterizing cellular and sub-cellular structures in vivo for coronary artery disease diagnosis.

We developed a new dual-modality intravascular imaging system based on fast time-gated fluorescence intensity imaging and spectral domain optical coherence tomography (SD-OCT) for the purpose of interventional detection of atherosclerosis. A pulsed supercontinuum laser was used for fluorescence and OCT imaging. A double-clad fiber (DCF)- based side-firing catheter was designed and fabricated to have a 23 μm spot size at a 2.2 mm working distance for OCT imaging. Its single-mode core is used for OCT, while its inner cladding transports fluorescence excitation light and collects fluorescent photons. The combination of OCT and fluorescence imaging was achieved by using a DCF coupler. For fluorescence detection, we used a time-gated technique with a novel single-photon avalanche diode (SPAD) working in an ultra-fast gating mode. A custom-made delay chip was integrated in the system to adjust the delay between the excitation laser pulse and the SPAD gate-ON window. This technique allowed to detect fluorescent photons of interest while rejecting most of the background photons, thus leading to a significantly improved signal to noise ratio (SNR). Experiments were carried out in turbid media mimicking tissue with an indocyanine green (ICG) inclusion (1 mM and 100 μM) to compare the time-gated technique and the conventional continuous detection technique. The gating technique increased twofold depth sensitivity, and tenfold SNR at large distances. The dual-modality imaging capacity of our system was also validated with a silicone-based tissue-mimicking phantom.

Stroke is one of the primary manifestations of cardiovascular disease affecting 15 million people annually worldwide. The bifurcation area of carotid arteries is particularly prone to plaque development, which is most often associated with ischemic strokes. Thus, early non-invasive detection of plaque vulnerability can have many positive implications for stroke prevention and healthcare costs. Optoacoustic imaging is increasingly finding applicability in clinical diagnostics, owing to its high resolution capacities and excellent label-free contrast related to vascular anatomy and haemodynamic deep within tissues. In this work, we developed a dedicated hand-held optoacoustic imaging probe for non-invasive imaging of the carotid artery and evaluate the feasibility of real-time volumetric imaging of the human carotids in vivo. The rapid volumetric image acquisition enables visualising the entire bifurcation area with 200µm spatial resolution free of motion artefacts e.g. related to breathing or pulsation. Carotid arteries located at depths between 5-15 mm from the skin surface were measured and their spectral contrast was assessed at varying excitation wavelengths in the 730-900nm range. Cross-sectional anatomical views of the bifurcation area were analysed in sagittal and transverse planes and compared with pulse-echo ultrasonography images. Volumetric image rendering in real time further enabled analysing the diameter and motion dynamics of the pulsing artery during cardiac cycle. The newly discovered capacity to non-invasively visualize the entire carotid bifurcation in three dimensions and real time may provide new insights on the structure and function of the human carotid artery toward reliable non-invasive detection of stenosis and plaque vulnerability.

Langendorff perfused hearts have been frequently studied in recent years using optical fluorescence imaging. This in vitro approach, which enables the heart to continue beating after extraction from the body of the animal, allows investigation of physiological functions with relative simplicity compared to in vivo setups. For example, when combined with voltage- and calcium- sensitive dyes, optical mapping of transmembrane potential, calcium transients, and other parameters can lead to a better understanding of cardiac mechanisms underlying heart failures and diseases. However, biomedical optical imaging is fundamentally limited to superficial investigations due to light scattering in tissues, restricting mapping to the heart surface only. The ability to visualize the heart septum would be important for comprehensive cardiac research. While 3D ultrasound can offer imaging of the entire heart, it can only provide mechanical contrast and the spatio-temporal resolution is also insufficient for imaging the heart in 3D on a beat-by-beat basis. Herein, we investigate on the capabilities of optoacoustic tomographic imaging of the Langendorff heart. The heart isolation method allows direct imaging without the presence of surrounding tissues and reduced blood content, significantly improving the penetration depth as well as image quality. The imaging system can acquire 3D images of the heart with optical contrast at an imaging rate of 100 Hz and 150 µm resolution. This enables capturing beat-by-beat heart motion with temporal resolution of 33 sampling instances per heartbeat. The high spatial resolution also allows identifying important internal heart features, including the septum, valves, cordae tendineae, and papillary muscles.

Subcellular resolution is required for OCT to portray the microstructural information of myocardium issue that is comparable to histology. Compare with its intrinsic intensity contrast, functional OCT system may provide contrast related to the tissue composition. We present a high-resolution (HR) cross-polarization OCT system that can provide functional contrast of human myocardium tissue in one-shot measurement. The system is implemented based on our previously reported high-resolution long imaging range OCT system with minimal modification. It features a broadband supercontinuum source, single-channel and one-shot detection, with moderate signal processing. The system has an axial resolution of 3.07 μm, and it is capable to produce accurate polarization information by calibrating the reconstruction performance with a quarter wave plate. The orthogonal polarization channels are multiplexed to fit within one imaging range. Following CP-OCT detection, the retardation can be reconstructed based on the complex signals, and the depolarization effect can be depicted by the channel intensity ratio. Tissue specimens from ten fresh human hearts are used to demonstrate the capability of CP-OCT contrasts. By analyzing the intrinsic and functional OCT contrasts of fresh human myocardium tissues against histology slides, we show that various tissue structures and tissue types of the myocardium, such as fibrosis and ablated lesions, can be better depicted by the function contrasts. We also suggest the possibility of using A-line features from the two orthogonal polarization channels to distinguish normal myocardium, fibrotic myocardium, and ablated lesions. This may serve as a rapid and cost-efficient solution for assessment of myocardium and further facilitate automatic tissue classification.

Remodeling processes associated with genetic and non-genetic cardiac diseases can cause alterations of electrical conduction and electro-mechanical dysfunction, eventually leading to arrhythmias. These alterations consist mainly in collagen deposition (fibrosis) and cellular disorganization (myofilament alignment), and their predictive models are often based on non-integrated and low-resolution information. Here, we combine advances in tissue clearing, immunostaining and high-resolution optical microscopy to reconstruct the three-dimensional organization of cardiac conduction system in the whole mouse heart. We developed a passive 2’2-thiodiethanol - Clarity protocol for clearing the heart and for achieving antibody penetration into the whole tissue. We simultaneously reconstructed the cellular organization stained by immunostaining and imaged the collagen distribution by second-harmonic generation deep in the cardiac tissue. A cytoarchitectonic analysis was applied to identify cells and to map myofilaments alignment in three dimensions, defining the conduction pathway of action potential propagation at intercellular level. We investigated the three-dimensional cytoarchitectonic remodeling in a transgenic mouse model of hypertrophic cardiomyopathy characterized by a severe degree of left ventricle hypertrophy and interstitial fibrosis. First, using a recently-developed ultra-fast optical system we mapped the propagation of electrical activity in whole diseased hearts. Then, we correlated the propagation maps with the pathological disorganization of myofilaments and collagen deposition using the three-dimensional high-resolution optical reconstruction. This innovative experimental approach will allow to dissect the morphological causes leading to alterations of electrical conduction and to electro-mechanical dysfunction, and, more generally, will represent a whole new paradigm for diagnostic and therapeutic investigations.

Aging is accompanied by complex structural changes in the heart. To explore this remodeling, we used a serial optical coherence tomography scanner to image the entire heart at a microscopic resolution. The imaging platform combines optical coherence microscopy to vibratome sectioning to automatically image every subsection of the sample. Post-processing algorithms were then used to stitch back together the sample in a large 3D dataset. We imaged the heart of 7 young (4 months) and 5 old (24 months) wildtype mice (C57B16) with the imaging platform. Optical coherence tomography of the myocardium reveals myofiber orientation that changes linearly from the endocardium to the epicardium. This change in orientation also varies with the distance from the apex of the heart : close to the apex, the change in myofiber orientation with respect to wall depth is larger. In old mice, this change was lower when compared to young mice due to remodelling. As reported in other works, the average volume of old mice hearts (97 ± 3 mm3) was significantly larger (p<0.05) when compared to young hearts (87 ± 3 mm3). Myocardial wall thickening was accompanied by a reduction of light attenuation in the endocardium. Attenuation coefficient in old mice endocardium was measured at 15.4 ± 0.4 cm-1, compared to 18.6 ± 0.5 cm-1 in young mice, which was significantly lower (p<0.05). The use of a serial optical coherence tomography allows new insight into fine changes of the whole heart.

Cardiac arrhythmias are a major source of mortality in the United States. Ablation is the only curative therapy for cardiac arrhythmias, and catheter-based radiofrequency ablation (RFA) through percutaneous access has emerged as the standard care for many arrhythmias. However, current procedures only monitor temperature, impedance and pressure measurements during ablation, which results in incomplete lesion formation (e.g., high recurrence rate for atrial fibrillation patients) and complications. In our previous work, we have shown integration of a commercially available RFA catheter with polarization sensitive optical coherence tomography (PSOCT). This was accomplished by housing the PSOCT probe inside of the RFA catheter with a window at the catheter tip that allows for forward viewing PSOCT imaging. Data from ex vivo experiments have shown that the integrated RFA catheter can be used to confirm catheter tissue apposition and monitor tissue change during ablation by measuring retardance. It was also demonstrated that the PSOCT window does not interfere with normal lesion formation. However, only the catheter tip was integrated, enabling only in vitro experiments. Therefore, we have developed a fully integrated PSOCT RFA catheter based on a commercially available catheter to enable experiments in living swine via percutaneous access. By properly choosing a middle layer sheath to house the PSOCT probe, we reduced the non-uniform rotation distortion (NURD), reduced PSOCT probe tip longitudinal movement due to winding back, improved image quality and stability. Further validation of functionality by simulating RFA procedures in living swine through percutaneous access is ongoing.

The gold standard of current treatment for atrial fibrillation is radiofrequency ablation (RFA). Single RFA procedures have low long-term, single-procedure success rates, which can be attributed to factors including inability to measure and visualize lesion depth in real time and incomplete knowledge of how atrial fibrillation manifests and persists. One way to address this problem is to develop a heart model that accurately fits lesion dimensions and depth using OCT to extract structural information. Twenty-three lesions of varying transmurality in left and right swine atrial tissue have been imaged with a Thorlabs OCT system with 6.5-micron axial resolution and a custom Ultra High Resolution system with 2.5-micron axial resolution. The boundaries of the ablation lesions were identified by the appearance of the birefringence artifact to identify areas of un-ablated tissue, as well as by changes to depth penetration and structural features, including decreased contrast between the endocardium and myocardium and disappearance of collagen fibers within the ablation lesion. Using these features, the lateral positions of the lesion boundaries were identified. An algorithm that fit ellipses to the lesion contours modeled the ablation geometry in depth. Lesion dimensions and shape were confirmed by comparison with trichrome histological processing. Finite-element models were fitted with these parameters and electrophysiological simulations were run with the Continuity 6 package. Next steps include correlating lesion geometry to conduction velocity, and including further tissue complexity such as varying tissue composition and fiber orientation. Additional models of linear lesions with gaps and adjacent lesions created with non-perpendicular contact will be created. This work will provide insight into how lesion geometry, tissue composition, and fiber organization impact electrophysiological propagation.

We studied the dependence of a perpendicular pressure to the luminal surface on the heating drug delivery performance using a laser-mediated thermal balloon with porcine carotid artery walls ex vivo. We proposed the combination use of our laser-mediated thermal balloon and drug coated balloon (DCB) to enhance a drug delivery performance. We prospected that the perpendicular pressure, which was applied directly to the luminal surface by balloon dilatation, would enhance the quantity of DCB drug delivered into artery wall. To simulate our laser thermal balloon heating, 63°C preheated artery samples were prepared by heated saline dropping for 15 s, and then these samples were dipped in 37°C saline for 15 s. Non-heated artery samples were prepared by dipping in 37°C saline for 30 s. The perpendicular pressure up to 10 atm corresponding to DCB dilatation pressure was added directly to these artery samples by fluorescence Rhodamine B solution for 30 s. The quantity of drug delivered was microscopically measured with fluorescence brightness in the cross-section of the drug delivered artery samples. We found drastic drug delivery increase at 8 atm using the pre-heated artery sample. Delamination of intima layer was observed by EVG stained cross-sectional specimens with 8 atm in the pre-heated artery sample. We think this drastic pressure dependence on the heating drug delivery performance might be corresponding to increase in permeability of drug into the artery wall originated to morphology change in intima.

Pulmonary vein (PV) isolation is a critical procedure for the treatment and termination of atrial fibrillation (AF). The success of such treatment depends on the extent of tissue damage, where partial lesions can allow abnormal electrical conduction and risk relapse of AF. Proper evaluation of lesion delivery and ablation line continuity remains challenging with current techniques and in part limit procedural efficacy. A tool for direct visualization of endo-myocardial lesions in vivo could potentially reduce ambiguity in treatment location and extent and improve the overall fidelity of lesion sets. In this work, we introduce a method for wide-field visualization of myocardial tissue including the discernment of ablated and non-ablated regions using an endoscopic multispectral imaging system (EMIS). The system was designed to fit the working channel of most commercial sheathes (<4 Fr) and supported quadruple-wavelength reflectance imaging through a flexible fiber-bundle. A total of 50 endocardial lesions were created and imaged on nine swine hearts, ex vivo in addition to 15 lesions on human LA samples near PV regions. A pixel-wise linear discriminant analysis algorithm was developed to classify regions of ablation treatment based on calibrated EMI maps. Results show good agreement of treatment severity and spatial extent compared to post-hoc tissue vital staining.

Several cardiovascular disease models for studying plaque rupture have been explored ranging from cell lines to animal models, whereby each one has contributed in different ways to pathology understanding, diagnostics and therapy. However, the scientific community is lacking a reliable animal model of human coronary plaque rupture. The model providing perhaps the highest degree of similarity to the human condition is the swine model that is known to exhibit serious disadvantages such as costly maintenance, time-consuming experiments, lasting several years and very low yield. Hence, we have developed a biomimetic artery modular platform where ex vivo human samples are exposed to an in vivo dynamic environment mimicking blood flow in order to get a rapid and reliable assessment of the intravascular diagnostic tool as well as to perform a multi-parametric screening with light. Our tool offers a unique environment to perform ex vivo dynamic studies such as dynamic biomarkers labelling tests, drug delivery, etc. The platform features computer-controlled experimental conditions and adjustable flow rates across the entire physiological range. Biological samples can be analyzed using traditional microscopy and assays or prepared for more advanced characterization after exposure in the test loop. Contrary to cell line models, the use of real artery samples provides a more comprehensive approach closer to human physiology. Interestingly, this approach allows remarkable time and resource savings compared to existent animal model of plaque vulnerability. Moreover, it offers higher flexibility on the biosample and mitigates related ethical issues.

Intravascular ultrasound (IVUS) imaging probes can be invaluable for guiding minimally invasive procedures such as coronary stent placement. With current IVUS catheters, ultrasound is generated and received electrically. With electronic transducer elements, it is challenging to achieve wide bandwidths, high sensitivity, and small dimensions suitable for intracoronary imaging. Here we present an all-optical ultrasound (OpUS) transducer, which uses light within fibre-optics to generate and receive ultrasound. These devices have several distinguishing advantages, including the potential to generate and receive wideband ultrasound (tens of MHz) required for high resolution imaging. The side-viewing OpUS transducer is highly miniaturised (< 1.5 mm diameter) with two optical fibres for transmission and reception, and a rotational mechanism for circumferential imaging. The transmitter is a composite of carbon nanotubes and PDMS coated on a multimode fibre tip. Ultrasound is generated within this coating by the photoacoustic effect. The receiver comprises a concave Fabry-Pérot cavity on a single mode fibre tip. Images acquired with the OpUS transducer were characterised using wire phantoms and post-mortem vascular tissue with stents. The axial resolution of this device was less than 70 microns, and the sensitivity was found to be sufficient to resolve pathological features. Subsequently, imaging was conducted in a healthy swine model in vivo and pulsatile motions of the artery were visualised with high fidelity. These studies show the strong potential for all-optical ultrasound to guide minimally invasive surgery.

Background: Birefringent crystals such as cholesterol and monosodium urate have recently been identified as possible pharmacologic targets for the treatment of coronary artery disease. The size of these crystals can be very small (on the order of 1 µm), making them difficult to identify. To image these microscopic crystals and enhance contrast, we modified existing micro optical coherence tomography system so that it was capable of obtaining polarization-sensitive images (PS-µOCT). A spectrometer-based PS-µOCT system was developed using a 270 nm wide broadband light source centered at 765 nm. Light was polarized using a polarizer and coupled to a SMF. The polarized light after SMF was divided into reference and sample arms using a beam splitter. Images of orthogonal polarization states were acquired sequentially by inserting and removing a quarter wave plate in the reference arm. The orthogonal PS- µOCT image components were used to generate birefringent images of the tissue. The axial resolution of the PS-µOCT system was 1.9 mm and the lateral resolution was 2 microns and the SNR was 92 dB. PS-µOCT was able to clearly identify isolated cholesterol and uric acid crystals. When used to image cadaver coronary arteries, the PS-µOCT images of crystals had up to 11 dB improved contrast compared to images obtained with a standard µOCT system. Results show that the use of PS-µOCT improves image contrast for isolated crystals and crystals within coronary atherosclerotic plaque and suggest that it could be useful for understanding their roles in the development and progression of coronary artery disease.

The extracellular matrix (ECM) plays crucial role in defining mechanical properties of a heart valve yet the mechanobiological role of the ECM proteins – collagen and elastin - in living heart valve leaflets is still poorly understood. In this study, non-linear microscopy was used to obtain three dimensional images of collagen and elastin arrangement in aortic leaflets under combined steady flow (850 ml/min) and cyclic flexure (1 Hz) mechanical (dynamic) training. A novel bioreactor capable of mimicking the flow conditions in a living heart was used in this study and was optimized for microscopic imagery. A custom made non-linear microscope was used in this study to provide Second Harmonic Generation (SHG) imaging of collagen arrangement and two-photon imaging of elastin. Two control and three trained leaflet samples from static and dynamic tissue culture were imaged to observe protein changes in the tissue for a period of seven days. Dynamic training led to a decrease in alignment index of the protein fibers compared to the static treatment.

All higher developed organisms contain complex hierarchical networks of arteries, veins and capillaries. These constitute the cardiovascular system responsible for supplying nutrients, gas and waste exchange. Diseases related to the cardiovascular system are among the main causes for death worldwide. In order to understand the processes leading to arteriovenous malformation, we studied hereditary hemorrhagic telangiectasia (HHT), which has a prevalence of 1:5000 worldwide and causes internal bleeding. In zebrafish, HHT is induced by mutation of the endoglin gene involved in HHT and observed to reduce red blood cell (RBC) flow to intersegmental vessels (ISVs) in the tail due to malformations of the dorsal aorta (DA) and posterior cardinal vein (PCV). However, these capillaries are still functional. Changes in the blood flow pattern are observed from in vivo data from zebrafish embryos through particle image velocimetry (PIV). Wall shear rates (WSRs) and blood flow velocities are obtained non-invasively with millisecond resolution. We observe significant increases of blood flow velocity in the DA for endoglin-deficient zebrafish embryos (mutants) at 3 days post fertilization. In the PCV, this increase is even more pronounced. We identified an increased similarity between the DA and the PCV of mutant fish compared to siblings, i.e., unaffected fish. To counteract the reduced RBC flow to ISVs we implement optical tweezers (OT). RBCs are steered into previously unperfused ISVs showing a significant increase of RBC count per minute. We discuss limitations with respect to biocompatibility of optical tweezers in vivo and determination of in vivo wall shear stress (WSS) connected to normal and endoglin-deficicent zebrafish embryos.

Intravascular photoacoustic/ultrasound imaging (IVPA/US) can image the structure and composition of atherosclerotic lesions identifying lipid-rich plaques ex vivo and in vivo. In the literature, multiple IVPA/US catheter designs were presented and validated both in ex-vivo models and preclinical in-vivo situations. Since the catheter is a critical component of the imaging system, we discuss here a catheter design oriented to imaging plaque in a realistic and translatable setting. We present a catheter optimized for light delivery, manageable flush parameters and robustness with reduced mechanical damage risks at the laser/catheter joint interface. We also show capability of imaging within sheath and in water medium.