Confocal microscopy and three-dimensional reconstruction of thick, transparent, vital tissue.

The three-dimensional visualization of the 400 micron thick, transparent, in situ cornea is described to demonstrate the use of confocal light microscopy for noninvasive imaging of living cells and thick tissues in their normal, vital conditions. Specimen preparation and physiological stability, as well as light attenuation corrections are critical to data acquisition. The technique to provide mechanical stability of the specimen during the duration of the image acquisition is explained. A laser scanning confocal light microscope (LSCM) was used to obtain optical serial sections from rabbit eyes that were freshly removed and placed in a physiological Ringer's solution. This study demonstrates the capability of the confocal light microscope to obtain a series of high contrast images, with a depth resolution of one micron, across the full thickness of living, transparent tissue. The problems of nonisotropic sampling and the limited eight-bit dynamic range are discussed. The three-dimensional reconstructions were obtained by computer graphics using the volume visualization projection technique. The three-dimensional visualization of the cornea in the in situ eye is presented as an example of image understanding of thick, viable biological cells and tissues. Finally, the criterion of image fidelity is explained. The techniques of confocal light microscopy with its enhanced lateral and axial resolution, improved image contrast, and volume visualization provides microscopists with new techniques for the observation of vital cells and tissues, both in vivo and in vitro.