Angular Orientation and Position of Lamella fibers, Derived from Second Harmonic Generation Microscopy Imaging

Abstract

The cornea and its parts, with particular focus on the corneal stroma, have developed into objects of great interest within the past few dozen years. By understanding the makeup of the corneal stroma in greater detail, we will better understand the structure and interaction of the cornea as a unit, as well as how the constituent components operate together. Our approach involved the use of second harmonic generation (SHG) microscopy, used to image several corneal stroma samples, in order to determine how the stromaÍs fibers were layered and oriented. This data was then processed using an algorithm we developed, which determined the angular orientation of the fibers in specific layers normal to the optical axis. As previous work utilized manual selection and subjective visual determination of the orientation, in order to determine the various orientations of fibers within corneal structures, the method presented here not only simplifies this process but is more accurate and robust. Our optical technique provides results that are consistent with more established techniques used at the lamella level, such as x-ray diffraction. æIn order to correct for possible displacement of a sample when imaging different depths, a correction algorithm processed and aligned each layer with the previously imaged layer. This way, a pixel in location (1,3,1) in layer A, matched up with a pixel in the same X-Y location (1,3,0) in layer B, so that cross sections could be stacked to model a three dimensional image of the sample.