Meng Han

The intensive metabolism of photoreceptors is delicately maintained by the retinal pigment epithelium (RPE) and the choroid. Dysfunction of either the RPE or choroid may lead to severe damage to the retina. Two-photon excited autofluorescence (TPEF) from endogenous fluorophores in the human retina provides a novel opportunity to reveal age-related structural abnormalities in the retina-choroid complex prior to apparent pathological manifestations of age-related retinal diseases. In the photoreceptor layer, the regularity of the macular photoreceptor mosaic is preserved during aging. In the RPE, enlarged lipofuscin granules demonstrate significantly blue-shifted autofluorescence, which coincides with the depletion of melanin pigments. Prominent fibrillar structures in elderly Bruch's membrane and choriocapillaries represent choroidal structure and permeability alterations. Requiring neither slicing nor labeling, TPEF imaging is an elegant and highly efficient tool to delineate the thick, fragile, and opaque retina-choroid complex, and may provide clues to the trigger events of age-related macular degeneration.

Nonlinear laser scanning microscopy is widely used for noninvasive imaging in cell biology and tissue physiology. However, multiphoton fluorescence imaging of dense, transparent connective tissue (e.g., cornea) is challenging since sophisticated labeling or slicing is necessary. High-resolution, high-contrast second harmonic generation (SHG) imaging of corneal tissue based on the intrinsic structure of collagen is discussed. The three-dimensional corneal ultrastructure in depths up to hundreds of microns can be probed noninvasively, without any staining or mechanical slicing. As an important application of second harmonic imaging in ophthalmology, the modification of corneal ultrastructure using femtosecond laser intrastromal ablation is systematically investigated to evaluate next-generation refractive surgical approaches. © 2004 Society of Photo-Optical Instrumentation Engineers.