Abstract. Label-free imaging of bulk arterial tissue is demonstrated using a multimodal nonlinear optical microscope based on a photonic crystal fiber and a single femtosecond oscillator operating at 800 nm. Colocalized imaging of extracellular elastin fibers, fibrillar collagen, and lipid-rich structures within aortic tissue obtained from atherosclerosis-prone myocardial infarction-prone Watanabe heritable hyperlipidemic ͑WHHLMI͒ rabbits is demonstrated through two-photon excited fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering, respectively. These images are shown to differentiate healthy arterial wall, early atherosclerotic lesions, and advanced plaques. Clear pathological changes are observed in the extracellular matrix of the arterial wall and correlated with progression of atherosclerotic disease as represented by the age of the WHHLMI rabbits. Atherosclerosis is the primary cause of heart disease, stroke, and lower limb amputation worldwide. It is a progressive disease characterized by chronic inflammation of injured intima and is associated with fatty plaque deposits in the arteries.1,2 Early atherosclerosis cannot be reliably detected by current clinical methods, therefore the disease is often overlooked until at a more advanced stage. The development of new tools that provide greater sensitivity and specificity for early detection and differentiation of atherosclerotic plaques would help our understanding of early disease and help establish preventative regimens that would slow disease progression.Recently, nonlinear optical ͑NLO͒ microscopy has emerged as a powerful tool for tissue imaging. It is a labelfree method with high sensitivity and specificity for major extracellular molecules. Its optical sectioning capability presents a means of 3-D in vivo imaging that would be useful in the context of atherosclerosis diagnostics. Several studies have demonstrated imaging of arterial tissue using NLO microscopy, [3][4][5][6] including studies imaging atherosclerotic lesions using a multimodal coherent anti-Stokes Raman Scattering ͑CARS͒ microscope based on two tightly synchronized Ti:sapphire lasers and a swine animal model. 6,7 In our study, we demonstrate label-free visualization of the extracellular matrix of arterial lumen and atherosclerotic plaques using a photonic crystal fiber ͑PCF͒-based multimodal NLO microscope employing only a single femtosecond oscillator. Twophoton excited autofluorescence ͑TPEF͒ is able to specifically image extracellular elastin fibers, second harmonic generation ͑SHG͒, type-1 collagen fibrils, and CARS lipid-rich structure or extracellular lipids droplets in unstained bulk intact tissue, indicating the methods are particularly suited to understanding the role and interplay between these key extracellular molecules involved in plaque development.PCF-based CARS was recently reported as an alternative CARS imaging method in biology. 8,9 Because it only requires a single femtosecond laser, PCF-based CARS can be easily integrated into existing...
A femtosecond CARS-based nonlinear optical microscope was used to simultaneously image extracellular structural proteins and lipid-rich structures within intact aortic tissue obtained from myocardial infarction-prone Watanabe heritable hyperlipidemic rabbits (WHHLMI). Clear differences in the NLO microscopic images were observed between healthy arterial tissue and regions dominated by atherosclerotic lesions. In the current ex-vivo study, we present a single parameter based on intensity changes derived from multi-channel NLO image to classify plaque burden within the vessel. Using this parameter we were able to differentiate between healthy regions of the vessel and regions with plaque, as well as distinguish plaques relative to the age of the WHHLMI rabbit.
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Structural proteins like collagen and elastin are major constituents of the extracellular matrix (ECM). ECM degradation and remodeling in diseases significantly impact the microorganization of these structural proteins. Therefore, tracking the changes of collagen and elastin fiber morphological features within ECM impacted by disease progression could provide valuable insight into pathological processes such as tissue fibrosis and atherosclerosis. Benefiting from its intrinsic high-resolution imaging power and superior biochemical specificity, nonlinear optical microscopy (NLOM) is capable of providing information critical to the understanding of ECM remodeling. In this study, alterations of structural fibrillar proteins such as collagen and elastin in arteries excised from atherosclerotic rabbits were assessed by the combination of NLOM images and textural analysis methods such as fractal dimension (FD) and directional analysis (DA). FD and DA were tested for their performance in tracking the changes of extracellular elastin and fibrillar collagen remodeling resulting from atherosclerosis progression/aging. Although other methods of image analysis to study the organization of elastin and collagen structures have been reported, the simplified calculations of FD and DA presented in this work prove that they are viable strategies for extracting and analyzing fiber-related morphology from disease-impacted tissues. Furthermore, this study also demonstrates the potential utility of FD and DA in studying ECM remodeling caused by other pathological processes such as respiratory diseases, several skin conditions, or even cancer. NEW & NOTEWORTHY Textural analyses such as fractal dimension (FD) and directional analysis (DA) are straightforward and computationally viable strategies to extract fiber-related morphological data from optical images. Therefore, objective, quantitative, and automated characterization of protein fiber morphology in extracellular matrix can be realized by using these methods in combination with digital imaging techniques such as nonlinear optical microscopy (NLOM), a highly effective visualization tool for fibrillar collagen and elastic network. Combining FD and DA with NLOM is an innovative approach to track alterations of structural fibrillar proteins. The results illustrated in this study not only prove the effectiveness of FD and DA methods in extracellular protein characterization but also demonstrate their potential value in clinical and basic biomedical research where protein microstructure characterization is critical.
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