Combination of digital image analysis and polarization microscopy: Theoretical considerations and experimental data

The potentialities of polarization microscopy has been greatly increased by using specific stains for selective enhancement of the optical anisotropy of a macromolecular constituent of cells and tissues. Such stainings have p roved to be especially useful in exploring the spatial orientation pattern of the extracellular matrix components. The retardation value, which charac terizes quantitatively the degree of submicroscopic orientation, can be mea sured traditionally with a compensator plate. This technique, however, is t ime-consuming and greatly dependent on visual judgment. Several attempts ha ve been made to combine digital image analysis and polarization microscopy to improve the measuring technique in unstained structures. In this paper, we summarize theoretical considerations and experimental data to show the a dvantages and limitations Of this methodological approach when using staine d and birefringent specimens. The technique we are suggesting is the measur ement of the light intensity using a 12 bit cCCD camera attached to a polar ized light microscope and digital image analysis system. The theoretical ba sis is given by the Fresnel equation describing the relationship between li ght intensity and retardation value. According to this, there is a sin(2) f unction between the light intensity and the retardation value. The same rel ationship of these two parameters was observed in our experiments on the bi refringent extracellular matrix around chondrocytes grown in agarose gel an d interterritorial and territorial matrix of canine articular cartilage sta ined with picrosirius red. Our results suggest that the retardation values can be calculated directly from the light intensity values if the retardati on value is lower than lambda/2. (C) 1998 Wiley-Liss, Inc.