Lipid distribution in human coronary lesions: analysis by digital imaging microscopy

Abstract

The pathophysiology of atherosclerosis occurs at the cellular level. Using a digital imaging fluorescence microscopy system and computer based methods, the 3-dimensional coordinates for lipid droplets, smooth muscle cells and macrophages in an arterial wall specimen can be obtained by a single experimental protocol. The experimental approach is to acquire at 2 μm intervals, bright field images and fluorescence images using a combination of lipid stains and monoclonal antibodies. After appropriate image processing, the data sets are combined to establish, with 3-dimensional coordinates, the spatial relationships of these components in the arterial wall. Quantitation of fluorescence requires information in three dimensions. It is obvious that the more easily observed two dimensional heterogeneity must also extend to three dimensions. The series of optical sections is usually achieved by moving the specimen along the z axis by computer controlled stage movement. When other fluorescent sources are nearby, but not in the focal plane, their fluorescence extend to overlap the fluorescent emission of the object of interest in the focal plane. The experimental problem of out-of-plane fluorescence is avoided by using a laser scanning confocal fluorescence microscope, which has an aperature in front of the detector to exclude fluorescence originating from locations other than the diffraction-limited excitation point source. We find predominately small intracellular nile red staining lipid droplets dispersed throughout the vessel wall. The larger extracellular droplets are confined largely to the lesion area, presumably in macrophages.