Quantitative Characterization Of Human Coronary Atherosclerotic Plaque Based On Multiphoton Microscopy

Coronary heart disease(CHD)caused by coronary atherosclerosis has been a leading cause of death worldwide.Coronary atherosclerotic plaque(CAP)rupture and resulting intracoronary thrombosis are thought to account for most acute coronary syndromes.Early identification and risk assessment of coronary atherosclerotic plaque is crucial for treatment selection to further decrease the incidence and mortality of coronary heart disease.Current diagnosing strategies such as clinical imaging methods are limited by low spatial resolution.As the gold standard,histopathological examination requires complicated and time-consuming sample preparation,and lack 3D imaging capabilities.Multiphoton microscopy(MPM)has the potential to circumvent this problem because it can achieve label-free,real-time,high resolution three dimensional imaging.It can provide the 3D structure of collagen and elastin fibers in coronary arterial intima at sub-cellular resolution.In this study,a home-built MPM imaging system with spectrum and lifetime detection function was used.A total of 43 regions from atherosclerotic plaque and normal region obtained from 9 human coronary arterial samples were imaged.810 nm excitation light was used to generate second harmonic generation(SHG)for collagen fibers and two-photon excited fluorescence(TPEF)signal for elastin fibers.At the same time,the structural and biochemical components information such as 3D morphology,spectrum and fluorescence lifetime of arterial intima were recorded.This article compares the CAP with normal region of human coronary artery intimal in biochemical composition and 3D microstructure,and explores potential quantitative indicators for distinguishing between diseased and normal intimal.MPM combined with 3D image analysis technology to achieve 3D microstructure quantitative analysis.Forty-three image stacks were analyzed by three dimensional gray level co-occurrence matrices(3D GLCM)and 3D weighted vector summation algorithm.Quantitative indicators were employed to describe the spatial microstructure alteration from normal and lesion tissues.We found that coronary atherosclerotic plaque tissues images exhibited spatial structural isotropy,asymmetry and intensity uniformity,disordered and blurred fibers compared with normal tissues.This study demonstrated that entropy,cluster shade,homogeneity and 3D directional variance of MPM images are capable of being 3D microstructure quantitative indicator for identifying the human coronary atherosclerotic lesions.MPM combined with spectroscopy and fluorescence lifetime detection technology to achieve biochemical composition quantitative analysis,according to spectrum,the spectral ratio between SHG and TPEF could be used to assess the content changes of collagen fibers and elastin fibers in coronary arterial wall.A stronger collagen fiber signal was found in the CAP regions.We also assessed the biochemical variations in coronary atherosclerotic tissues by measuring the fluorescence lifetime and found a lower mean fluorescence lifetime in coronary atherosclerotic plaque compared with normal tissue.This study demonstrated that the TPEF spectrum,TPEF/SHG and mean fluorescence lifetime are capable of being biochemical composition quantitative indicator for identifying the human coronary atherosclerotic lesions.