In this study we present a novel image analysis methodology to quantify and to classify morphological details in tissue collagen fibril organization and lipid deposition. Co-localized collagen (second harmonic, SHG) and lipid (coherent Raman, CARS) images of atherosclerotic artery walls were acquired by a supercontinuum-powered multi-modal nonlinear microscope. Textural features based on the first-order statistics (FOS) and gray level co-occurrence matrix (GLCM) parameters were extracted from the SHG and CARS images. Multi-group classifications based on support vector machine of SHG and CARS images were subsequently performed to investigate the potential of texture analysis in providing quantitative descriptors of structural and compositional changes during disease progression. Using a rabbit model, different collagen remodeling and lipid accumulation patterns in disease tissues can be successfully tracked using these image statistics, thus providing a robust foundation for classification. When the variation of the CARS image features were tracked against the age of the rabbit, it was noticed that older animals (advanced plaques) present a more complex necrotic core containing high-lipid extracellular structures with various shapes and distribution. With combined FOS and GLCM texture statistics, we achieved reliable classification of SHG and CARS images acquired from atherosclerotic arteries with >90% accuracy, sensitivity and specificity. The proposed image analysis methodology can also be applied in a wide range of applications to evaluate conditions involving collagen re-modeling and prominent lipid accumulation.
Luminal atherosclerosis imaging was demonstrated by multimodal femtosecond CARS
microscopy (MM-CARS). Using a myocardial infarction-prone rabbit model of
atherosclerosis, this study demonstrated the utility of multimodal CARS imaging in
determining atherosclerotic plaque burden through two types of image analysis procedures.
Firstly, multimodal CARS images were evaluated using a signal-intensity parameter based
on intensity changes derived from the multi-channel data (e.g. TPEF, SHG and CARS) to
classify plaque burden within the vessel. Secondly, the SHG images that mainly correspond
to collagen fibrils were evaluated using a texture analysis model based on the first-order
statistical (FOS) parameters of the image histogram. Correlation between FOS parameters of
collagen images with atherosclerosis plaque burden was established. A preliminary study
of using spectroscopic CARS in identifying the different lipid components within the plaque
was also discussed.
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 (WHHLMI) 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.
Nonlinear optical (NLO) microscopy provides a minimally invasive optical method for
fast molecular imaging at subcellular resolution with 3D sectioning capability in thick,
highly scattering biological tissues. In the current study, we demonstrate the imaging
of arterial tissue using a nonlinear optical microscope based on photonic crystal fiber
and a single femto-second oscillator operating at 800nm. This NLO microscope system
is capable of simultaneous imaging extracellular elastin/collagen structures and lipid
distribution within aortic tissue obtained from coronary atherosclerosis-prone WHHLMI
rabbits (Watanabe heritable hyperlipidemic rabbit-myocardial infarction) Clear
pathological differences in arterial lumen surface were observed between healthy
arterial tissue and atherosclerotic lesions through NLO imaging.