Improved label-free diagnostics and pathological assessment of atherosclerotic plaques through nonlinear microscopy

Coronary heart disease is the most common type of heart disease caused by atherosclerosis. In fact, an arterial wall lesion centered on the accumulation of cholesterol-rich lipids and the accompanying inflammatory response generates a plaque, whose rupture may result in a thrombus with fatal consequences. Plaque characterization for assessing the severity of atherosclerosis is generally performed through standard histopathological examination based on hematoxylin/eosin staining, which is operator-dependent and requires relatively long procedures. In this framework, nonlinear optical microscopy is a valid, label-free alternative to standard diagnostic methods. We combined second-harmonic generation (SHG), two-photon excited fluorescence (TPEF) and fluorescence lifetime imaging microscopy in a multimodal scheme for obtaining morphological and molecular information on human carotid ex vivo specimens affected by atherosclerosis. In this study, discrimination between different tissues within the atherosclerotic plaque was achieved based on both lifetime, TPEF-to-SHG ratio, and image pattern analysis. The presented methodology aims to be a starting point for future fully automated and fast characterization of atherosclerotic biopsies; moreover, it could be extended to the study of other tissues and pathologies. Combined TPEF/SHG mapping of a carotid specimen affected by atherosclerosis