Label-Free Quantitative Imaging of Cholesterol in Intact Tissues by Hyperspectral Stimulated Raman Scattering Microscopy

摘要

Cholesterol is an indispensable structural constituent of membranes, and it also plays a prominent role in the biosynthesis of steroid hormones. Excessive cholesterol storage is a hallmark of atherosclerotic cardiovascular diseases. Crystalline cholesterol has not only been found in advanced atherosclerotic lesions, but it has also been recognized as an inflammatory stimulus that appears in a very early stage of atherosclerosis. Abnormal storage of cholesterol also occurs in steroidogenic cells and Niemann-Pick type C disease. However, quantitative imaging of cholesterol and its esterified form in an intact biological tissue remains a challenge. The widely applied histological hematoxylin and eosin staining only allows indirect mapping of crystalline cholesterol clefts. Lipid-soluble Sudan dyes, such as Oil Red O and Bromine-Sudan black, are widely used to stain neutral lipid droplets that contain the cholesteryl ester and triglyceride, but these dyes neither stain crystalline cholesterol, nor elucidate the composition of lipid droplets. Filipin is a gold standard for staining free cholesterol in fixed cells, but is unable to label either large cholesterol crystals or lipid droplets that contain the cholesteryl ester. Confocal laser reflection microscopy and micro-optical coherence tomography have been successfully implemented to visualize reflective substances such as cholesterol and silica, but neither has the capability for chemical identification.