In vivo imaging of the human eye using a 2-photon-excited fluorescence scanning laser ophthalmoscope

BACKGROUND: Noninvasive assessment of metabolic processes that sustain regeneration of human retinal visual pigments (visual cycle) is essential to improve ophthalmic diagnostics and to accelerate development of new treatments to counter retinal diseases. Fluorescent vitamin A derivatives, which are the chemical intermediates of these processes, are highly sensitive to UV light; thus, safe analyses of these processes in humans are currently beyond the reach of even the most modern ocular imaging modalities. METHODS: We present a compact, 2-photon-excited fluorescence scanning laser ophthalmoscope and spectrally resolved images of the human retina based on 2-photon excitation (TPE) with near-infrared light. A custom Er:fiber laser with integrated pulse selection, along with intelligent postprocessing of data, enables excitation with low laser power and precise measurement of weak signals. RESULTS: We demonstrate spectrally resolved TPE fundus images of human subjects. Comparison of TPE data between human and mouse models of retinal diseases revealed similarity with mouse models that rapidly accumulate bisretinoid condensation products. Thus, visual cycle intermediates and toxic byproducts of this metabolic pathway can be measured and quantified by TPE imaging. CONCLUSION: Our work establishes a TPE instrument and measurement method for noninvasive metabolic assessment of the human retina. This approach opens the possibility for monitoring eye diseases in the earliest stages before structural damage to the retina occurs. FUNDING: NIH, Research to Prevent Blindness, Foundation for Polish Science, European Regional Development Fund, Polish National Agency for Academic Exchange, and Polish Ministry of Science and Higher Education.