Characterization of human atherosclerotic plaques using multimodal multiphoton microscopy

Atherosclerosis is a widespread cardiovascular disease caused by the deposition of lipids (such as cholesterol and triglycerides) on the inner arterial wall. It is one of the leading causes of death in the Western World, as the rupture of an atherosclerotic plaque may result in a thrombus with fatal consequences. Preventive assessment of plaque vulnerability is therefore extremely important and it could be performed by studying collagen organization and lipid composition in atherosclerotic arterial tissues. Nonlinear microscopy is a labelfree and non-invasive tool for achieving such goal, as an alternative to the histopathological examination. In this study, we used Second Harmonic Generation (SHG) microscopy, Two-Photon Fluorescence (TPF) microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) with a combined approach in order to characterize collagen organization and lipids in human carotid ex vivo tissue sections affected by atherosclerosis. SHG and TPF images, acquired from different regions within atherosclerotic plaques, were used to measure the corresponding TPF/SHG ratios. Moreover, such images were processed through image pattern analysis methods such as Fast Fourier Transform (FFT) and Grey-Level Co-occurrence Matrix (GLCM). Finally, the resulting information on collagen and cholesterol distribution and anisotropy, combined with collagen and lipids fluorescence lifetime measured from FLIM images, allowed characterizing carotid samples and discriminating different tissue regions. The presented method offers the potential to be applied for automated classification of atherosclerotic lesions and plaque vulnerability. In addition, the methodology could be extended to the diagnostics of other tissues and pathologies. Moreover, it lays the foundation for a potential in vivo diagnostic tool to be used in a clinical setting.