In vivo imaging of the human eye using a 2-photon-excited fluorescence scanning laser ophthalmoscope
Jakub Boguslawski, Grazyna Palczewska, Slawomir Tomczewski, Jadwiga Milkiewicz, Piotr Kasprzycki, Dorota Stachowiak, Katarzyna Komar, Marcin J. Marzejon, Bartosz L. Sikorski, Arkadiusz Hudzikowski, Aleksander Głuszek, Zbigniew Łaszczych, Karol Karnowski, Grzegorz Soboń, Krzysztof Palczewski, Maciej Wojtkowski
Jakub Boguslawski, Grazyna Palczewska, Slawomir Tomczewski, Jadwiga Milkiewicz, Piotr Kasprzycki, Dorota Stachowiak, Katarzyna Komar, Marcin J. Marzejon, Bartosz L. Sikorski, Arkadiusz Hudzikowski, Aleksander Głuszek, Zbigniew Łaszczych, Karol Karnowski, Grzegorz Soboń, Krzysztof Palczewski, Maciej Wojtkowski
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Clinical Research and Public Health Ophthalmology

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

Abstract

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.

Authors

Jakub Boguslawski, Grazyna Palczewska, Slawomir Tomczewski, Jadwiga Milkiewicz, Piotr Kasprzycki, Dorota Stachowiak, Katarzyna Komar, Marcin J. Marzejon, Bartosz L. Sikorski, Arkadiusz Hudzikowski, Aleksander Głuszek, Zbigniew Łaszczych, Karol Karnowski, Grzegorz Soboń, Krzysztof Palczewski, Maciej Wojtkowski

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Figure 4

TPEF provides safe fundus imaging without any structural or functional changes.

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TPEF provides safe fundus imaging without any structural or functional c...
(A) TPEF fundus image of subject 2 centered at the fovea. Total exposure: 163 seconds. (B–E) Data obtained before TPEF-SLO imaging; yellow arrows point to depigmentation region in all. (B) B-FAF image. (C) NIR-FAF image. (D) Visual field tested by perimetry. (E) OCT b-scan; red arrows indicate the spread of the depigmentation. (F) TPEF fundus image of subject 2 centered at 6.9° eccentricity nasally from the fovea; the yellow arrow points to an area with a clear hypofluorescent lesion. Total exposure: 162 seconds. (G–J) Data obtained 1 month after TPEF imaging. (G) B-FAF. (H) NIR-FAF. (I) OCT. (J) Perimetry. In A and F, 100 frames were averaged to generate the images.