3D Volume Reconstruction of Extracellular Matrix Proteins in Uveal Melanoma from Fluorescent Confocal Laser Scanning Microscope Images.

Peter Bajcsy, Sang-Chul Lee, Amy Y. Lin and Robert Folberg

Journal of Microscopy 221 p30-45 (2006)

The distribution of looping patterns of laminin in uveal melanomas and other tumors has been associated with adverse outcome. Moreover, these patterns are generated by highly invasive tumor cells through the process of vasculogenic mimicry and are not therefore blood vessels. Nevertheless, these extravascular matrix patterns conduct plasma. The three-dimensional configuration of these laminin-rich patterns compared with blood vessels has been the subject of speculation and intensive investigation. We developed a method for the three-dimensional reconstruction of volume for these extravascular matrix proteins from serial paraffin sections cut at 4 µm thicknesses and stained with a fluorescent-labeled antibody to laminin. Each section was examined with confocal laser-scanning focal microscopy (CLSM) and 13 images were recorded in the Z-dimension for each slide. The input CLSM imagery is composed of a set of 3D sub-volumes (stacks of 2D images) acquired at multiple confocal depths, from a sequence of consecutive slides. Steps for automated reconstruction process included (1) unsupervised methods for selecting an image frame from a sub-volume based on entropy and contrast criteria, (2) a fully automated registration technique for image alignment, and (3) an improved histogram equalization method that compensates for spatially varying image intensities in CLSM imagery due to photo-bleaching. We compared image alignment accuracy of a fully automated method with registration accuracy achieved by human subjects using a manual method. In this study, automated 3D volume reconstruction provided significant improvement in accuracy, consistency of results, and performance time for CLSM images acquired from serial paraffin sections.