Polarization-modulated second harmonic generation imaging: method for quantitative assessment of disorganization in anulus

An experimental method for quantifying disorder within the anulus fibrosus is described based on polarization-modulated second harmonic generation imaging (PM-SHG-I). This method is demonstrated by imaging the anular lamellar architecture of a mouse model of compressive loading. Results were consistent with those obtained in an earlier study where organization was quantified directed secants image analysis on photomicrographs. In this study the orientation within individual lamellia is quantified by average orientation of the collagen molecules within a defined volume of a single lamellar as measured by the PM-SHG-I. Lamellar boundaries can be identified through the SHG intensity images, and confirmed through co-registration with photomicrographs of the same region. The orientation within the lamellar is quantified by the polarization angle of the maximum second harmonic intensity. PM-SHG-I offers several advantages as compared with the method of directed secants: first, it is nondestructive, allowing repeated measurements of the same tissue; second, images are captured on the order of seconds and capable of obtaining information up to a depth of 200-300 microns, thus allowing for real-time assessment of load damage; third, organization is measured at a much higher resolution, as it is based on disorder within the molecular arrays of a single lamella.