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...
The objective of this study was to compare two noninvasive techniques, laser Doppler and optical spectroscopy, for monitoring hemodynamic changes in skin flaps. Animal models for assessing these changes in microvascular free flaps and pedicle flaps were investigated. A 2 x 3-cm free flap model based on the epigastric vein-artery pair and a reversed MacFarlane 3 x 10-cm pedicle flap model were used in this study. Animals were divided into four groups, with groups 1 (n = 6) and 2 (n = 4) undergoing epigastric free flap surgery and groups 3 (n = 3) and 4 (n = 10) undergoing pedicle flap surgery. Groups 1 and 4 served as controls for each of the flap models. Groups 2 and 3 served as ischemia-reperfusion models. Optical spectroscopy provides a measure of hemoglobin oxygen saturation and blood volume, and the laser Doppler method measures blood flow. Optical spectroscopy proved to be consistently more reliable in detecting problems with arterial in flow compared with laser Doppler assessments. When spectroscopy was used in an imaging configuration, oxygen saturation images of the entire flap were generated, thus creating a visual picture of global flap health. In both single-point and imaging modes the technique was sensitive to vessel manipulation, with the immediate post operative images providing an accurate prediction of eventual outcome. This series of skin flap studies suggests a potential role for optical spectroscopy and spectroscopic imaging in the clinical assessment of skin flaps.
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