High throughput en face mapping of arterial permeability using tile scanning confocal microscopy

Abstract

Elevated uptake of plasma macromolecules by the arterial wall has been implicated in the initiation of atherosclerosis. Here we describe a new method for mapping such uptake in laboratory animals. Albumin was labelled with a fluorescent dye and administered intravenously. After 10 min, the aorta was fixed in situ, excised and opened. En face confocal microscopy employing a computer-controlled stage was used to obtain contiguous tiles, each consisting of a stack of images of fluorescence emission at different depths in the wall. To obtain two-dimensional maps, intensities were summed in each column of voxels starting at the endothelial surface and extending 10 μm into the wall. Variation in the sensitivity of the system with time and in all three spatial directions was assessed and corrected using calibration standards and model specimens. In immature rabbits, uptake around aorto-intercostal branches was greatest in an arrowhead-shaped region around the downstream half of each ostium, and at its lateral margins. Uptake around branches in mature rabbits was more uniform; it was highest upstream of the ostium. Patches and streaks of high uptake were also seen at non-branch locations in the descending thoracic aorta. Transport was more uniform around branches in mice, except for small regions of high uptake at the ostial rim and at the leading edge of an intimal cushion upstream of the ostium, where lesions develop. The technique provides accurate quantification in three dimensions over large areas; it has high throughput, sensitivity and resolution and is suitable for widespread use.