In vivo imaging of the human eye using a 2-photon-excited fluorescence scanning laser ophthalmoscope
Jakub Boguslawski, … , Krzysztof Palczewski, Maciej Wojtkowski
Jakub Boguslawski, … , Krzysztof Palczewski, Maciej Wojtkowski
Published November 30, 2021
Citation Information: J Clin Invest. 2022;132(2):e154218. https://doi.org/10.1172/JCI154218.
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Clinical Medicine 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 1

Two-photon-excited fluorescence scanning laser ophthalmoscope (TPEF-SLO) driven by a femtosecond fiber laser.

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Two-photon-excited fluorescence scanning laser ophthalmoscope (TPEF-SLO)...
(A) Experimental setup of TPEF-SLO, including 4 major units: femtosecond laser, second harmonic generation (SHG) module, dispersion precompensation, and SLO module; inset represents image processing (each unit is described in detail in Methods). L, lens; GS, galvanometer-based x-y scanners; DM, dichroic mirror; BP, set of bandpass filters; PMT, photomultiplier tube; MMF, multimode fiber; APD, avalanche photodiode. (B) Retrieved pulse intensity and phase measured in the retinal plane. (C) Optical spectrum of the laser measured in the pupil plane. (D) Retinal exposure vs. exposure time (red curve = equivalent of MPE calculated for static beam case) and comparison of retinal exposures used in this study and in Schwarz et al. (24); adapted from Schwarz et al. (24) with with permission from The Optical Society of America. (E) Relative TPEF intensity as a function of pulse repetition frequency (PRF, black curve), illustrating the effect of reduced PRF. Shown is the calculated average excitation power (blue curve) needed to obtain the same fluorescence intensity as for 0.3 mW and 6 MHz used in this study. Red line shows MPE calculated for static beam case and 40-second exposure time.