We report the realization of vibrational photoacoustic (VPA) microscopy using optical excitation of molecular overtone vibration and acoustic detection of the resultant pressure transients. Our approach eliminates the tissue scattering problem encountered in near-infrared spectroscopy and enables depth-resolved signal collection. The 2nd overtone of the CH bond stretch around 8300 cm−1, where blood interference is minimal, is excited. We demonstrate 3D VPA imaging of lipid-rich atherosclerotic plaques by excitation from the artery lumen, and lipid storage in live Drosophila larvae, with millimeter-scale penetration depth.

We report the employment of an optical window between 1600 nm and 1850 nm for bond-selective deep tissue imaging through harmonic vibrational excitation and acoustic detection of resultant pressure waves. In this window where a local minimum of water absorption resides, we found a 5 times enhancement of photoacoustic signal by first overtone excitation of the methylene group CH2 at 1730 nm, compared to the second overtone excitation at 1210 nm. The enhancement allows 3D mapping of intramuscular fat with improved contrast and of lipid deposition inside an atherosclerotic artery wall in the presence of blood. Moreover, lipid and protein are differentiated based on the first overtone absorption profiles of CH2 and methyl group CH3 in this window. VPA imaging of atherosclerotic plaque with the presence of blood.

Objective-The purpose of this study was to assess the ability of label-free multimodal nonlinear optical (NLO) microscopy to characterize, and thus enable quantitative in situ analyses of, different atherosclerotic lesion types, according to the original scheme suggested by the AHA Committee. Methods and Results-Iliac arteries were taken from 24 male Ossabaw pigs divided into lean control and metabolic syndrome groups and were imaged by multimodal NLO microscopy where sum-frequency generation (SFG) and 2-photon excitation fluorescence (TPEF) were integrated on a coherent anti-Stokes Raman scattering (CARS) microscope platform. Foam cells, lipid deposits, matrices, and fibrous caps were visualized with submicron 3D resolution. Starting from the adaptive intimal thickening in the initial stage to the fibrous atheroma or mineralization in the advanced stages, lesions were visualized without labels. Histological staining of each lesion confirmed the lesion stages. Lipid and collagen contents were quantitatively analyzed based on the CARS and SFG signals. Lipid accumulation in thickened intima culminated in type IV whereas the highest collagen deposition was found in Type V lesions. Luminal CARS imaging showed the capability of viewing the location of superficial foam cells that indicate relatively active locus in a lesion artery. Key Words: coherent antistokes Raman scattering Ⅲ multimodal nonlinear optical microscopy Ⅲ Ossabaw miniature swine Ⅲ atherosclerosis Ⅲ histopathology A therosclerosis, the major cause of cardiovascular diseases, has been a leading contributor to morbidity and mortality in the United States, 1 and it has been on the rise globally. 2 The statistics that account for the rise in incidence consequently call for new imaging techniques to advance the research and diagnosis of atherosclerosis. Current imaging methods, such as x-ray angiography, MRI, intravascular ultrasound (IVUS), computed tomography, and optical coherence tomography (OCT) allow exquisite delineation of advanced lesions. 3 Notably, MRI can achieve molecular imaging using contrast agents. 4,5 Also, by using spectral analysis to identify lesion components, IVUS can perform virtual histology. 6 -8 Nonetheless, these techniques have not yet reached submicron resolution. As the gold standard, histology provides high performance in biopsy studies, but it is not feasible for live tissue imaging. In addition, with technical advances, fluorescence microscopy with 1-and 2-photon excitation has been applied to vascular and atherosclerosis studies 5,9 with the capability of identifying cellular and molecular compositions in vivo with labeling. 10 However, these techniques are subject to possible compromises to labeling, such as photobleaching, the requirement for extra incubation, or limited circulation lifetime, all of which could be less optimal for arterial studies. Thus, it is intriguing to explore label-free imaging methods which can also provide chemical selectivity and submicron resolution. Conclusions-WeNonlinear optical (NLO) m...

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