Mapping of the orientation of myocardial cells by means of polarized light and confocal scanning laser microscopy.

The study of the topological organisation of myocardial cells is a basic requirement for the understanding of the mechanical design of the normal and pathological heart. We developed a technique based on multiparametric image analysis of transmitted polarized light to generate maps of the azimuth and the elevation angles of the myocardial cells. The properties of birefringence of the myocardium embedded in methylmetacrylate were measured in papillary muscles with monitored 3D orientation. This birefringence is positive uniaxial with a 0 degree extinction angle when the axis of the fiber is parallel to the axis of the polarizer or the analyzer. Thick sections were studied between crossed polars, and four images of each section were digitized for an angle of the polarizer with the section varying from 0-67.5 degrees in steps of 22.5 degrees. The amounts of transmitted light for each setup of the polarizer were combined in order to extract the values of the azimuth angle (modulo 90 degrees) and the elevation angle of the myocardial cells, according to the Johannsen equation. The respective maps of these angles were calculated and then assessed with confocal scanning laser microscopy. This method provides an efficient and accurate tool for the study of the histological architecture of the fetal and neonatal heart.