Methods of achieving three-dimensional reconstruction of tissue at the ultrastructural level demonstrating the distribution of melanosomes within retinal pigment epithelium

摘要

In predicting and measuring laser effect on retinal tissue for most of the visible to near infrared spectrum, one is concerned with the melanosome as the major absorber of incident energy. Differences in the location and density of melanosomes in the retinal pigment epithelium may have an impact on the effect of laser energy delivered to those tissues. Current models use estimates of numbers of melanosomes usually in an even distribution across a 5 - 8 micrometer deep volume. The goal of our study is to identify the three-dimensional distribution of melanosomes within the retinal pigment epithelium (RPE) for the use of those modeling laser tissue effects. We examined normal retinal pigment epithelium using three-dimensional (3-D) reconstruction from images obtained by transmission electron microscopy (TEM), light microscopy (LM) and confocal microscopy. Images were captured on a digital camera system attached to the microscope for both the transmission electron and light microscopy. Three-dimensional reconstruction was performed after digital deconvolution of microscopic images (Vaytek, Inc.). Three- dimensional images were then utilized for analysis of distribution of melanosomes and organelles within the pigment epithelial block. The distribution of melanosomes will be useful for accurate mathematical modeling of laser impact on the retina.