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 2

TPEF-SLO enables imaging of endogenous fundus chromophores in eye of a healthy subject 1.

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TPEF-SLO enables imaging of endogenous fundus chromophores in eye of a h...
(A) TPEF-SLO image of the fundus centered at 7.7° eccentricity nasally from foveal region of interest (ROI) 1. Image obtained by averaging of 1000 frames in spectral window 400 to 700 nm. (B) TPEF-SLO image of the fundus centered at 2.5° eccentricity nasally from fovea, ROI 2. Image obtained by averaging 100 frames. (C) TPEF-SLO image from ROI 1 location acquired with continuous wave (CW) excitation at 825 nm, showing significantly decreased TPEF signal intensity. Image obtained by averaging 100 frames. (D–F) Confocal reflectance images corresponding to panels A–C. (G) Quantification of fluorescence intensity with respect to leakage of backscattered light and noise floor (dark counts and stray light) (n = 10, 5, and 10, respectively). The upper and lower bands indicate second and third quartiles, respectively; the line within the box indicates the mean value; whiskers extend to minimum and maximum values. (H) Normalized fluorescence as a function of eccentricity at ROI 2 compared with the corresponding region measured by B-FAF and NIR-FAF methods. (I) Confocal B-FAF image (488 nm excitation). (J) Confocal NIR-FAF image. In I and J, blue circles mark the location at 7.3° eccentricity nasally from fovea (ROI 1), and red circles mark the macular region (ROI 2). In B and E, circles outlined in dashed black dashed lines mark the location of fovea centralis. Scale bars: 1 mm.