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 3

Spectral properties of TPEF of human fundus compared with selected mouse models.

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Spectral properties of TPEF of human fundus compared with selected mouse...
(A) Human TPEF images of subject 1 at approximately 7.5° eccentricity nasally from fovea (ROI 1), recorded in spectral ranges of 594–646 nm, 400–600 nm, 500–540 nm, and 400–550 nm, and normalized to the image acquired for 400–700 nm; 1000 frames were used. (B) TPEF fundus images of albino (Alb.) Abca4–/– Rdh8–/– mice in vivo recorded in corresponding spectral ranges normalized to the TPEF image obtained in the 400–700 nm spectral range. (C) Plot showing relative fluorescence change in 4 spectral ranges normalized with respect to 400–700 nm for human TPEF imaging (n = 10). (D) Plot showing relative fluorescence change in 4 spectral ranges normalized with respect to 400–700 nm for 5 mouse models (n = 6, 4, 4, 4, and 4). Pigm., pigmented. *P > 0.2, ***P < 0.005 by 1-way ANOVA with Bonferroni’s post hoc test. (E) FLIM images of albino Abca4–/– Rdh8–/–, pigmented Abca4–/– Rdh8–/–, pigmented Rpe65–/–, and BALB/cJ mice. Red arrows point to retinosomes (57), and white arrows point to macrophages. (F) Phasor plots corresponding to data presented in panel E. In each universal semicircle, clusters of phasor points are color coded from blue to red, where red represents highest phasor point density. Color bars drawn through clusters of phasor points represent color scales for FLIM images in E. Yellow circles outline grouping of phasor points in albino and pigmented mouse RPE. Error bars represent SD.