Three-dimensional confocal microscopy of the living in situ rabbit cornea

Three-dimensional confocal microscopy of a living in situ rabbit cornea, in a freshly excised eye, is demonstrated in two movie loops. A specimen chamber for the eye was designed and constructed to maintain the unstained, unfixed, in situ cornea in a viable physiological state during data acquisition. The 400 micron thick, transparent, cornea has been optically sectioned into 365 sections using a laser scanning confocal microscope. A high numerical aperture, water immersion microscope objective minimized the spherical aberrations which would occur with the use of an oil immersion objective. Depth dependent light attenuation due to absorption and scatter within the specimen was manually compensated at each sampled section. 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.