3-DIMENSIONAL CONFOCAL MICROSCOPY AND VISUALIZATION OF THE IN-SITU CORNEA

The in situ cornea is an ideal test specimen to evaluate techniques for 3D reconstruction and visualization of unstained, unfixed, transparent living tissues from a stack of optical sections. The 0.4 mm thick transparent specimen has been optically sectioned into 365 sections using a confocal laser scanning microscope (CLSM) with a water immersion objective. Depth-dependent light attenuation due to absorption and scatter within the specimen was manually compensated at each sampled section. A water immersion microscope minimized the spherical aberrations that would have occurred with the use of an oil immersion objective. Isometric sampling resulted in near-cubic voxels, which compensated for the reduced microscopic resolution in the z axis as compared to x and y resolution.