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.

Two-photon vision is a phenomenon associated with the perception of short pulses of near-infrared radiation (900-1200 nm) as a visible light. It is caused by the nonlinear process of two-photon absorption by visual pigments. Here we present results showing the influence of pulse duration and repetition rate of short pulsed lasers on the visual threshold. We compared two-photon sensitivity maps of the retina obtained for subjects with normal vision using a cost-effective fiber laser (λ_c = 1028.4 nm, τ_p = 12.2 ps, F_rep = 19.17 MHz) and a solid-state laser (λ_c = 1043.3 nm, τ_p = 0.253 ps, F_rep = 62.65 MHz). We have shown that in accordance with the description of two-photon absorption, the average optical power required for two-photon vision for a fiber laser is 4 times greater than that for a solid-state laser. Mean sensitivity measured for the first one is 5.9 ± 2.8 dB lower than for the second but still 17 dB away from the safety limit, confirming that picosecond light sources can be successfully applied in microperimetry. This development would dramatically reduce the cost and complexity of future clinical devices.

We report the development of a widely-tunable femtosecond fiber laser system and its application for two-photon vision studies. The source is based on an Er-doped fiber laser with spectral shift up to 2150 nm, followed by a second harmonic generation module to generate a frequency-doubled beam tunable from 872 to 1075 nm. The source delivers sub-230 fs pulses with nearly-constant duration over the entire tuning range, with output powers between 0.68–1.24 mW, which corresponds to a pulse energy of 13.2–24.1 pJ. Such pulse energy is sufficient for employing a system for measurements of two-photon scotopic spectral sensitivity of two-photon vision in humans. The laser parameters allow for very efficient and safe two-photon stimulation of the human visual system, as proved by a good separation between one- and two-photon thresholds for wavelengths below 950 nm, which we have confirmed for 3 healthy subjects.

This report presents the results of measurements of the two-photon vision threshold for various pulse trains. We employed three pulsed near-infrared lasers and pulse stretchers to obtain variations of the pulse duty cycle parameter over three orders of magnitude. We proposed and extensively described a mathematical model that combines the laser parameters with the visual threshold value. The presented methodology enables one to predict the visual threshold value for a two-photon stimulus for a healthy subject while using a laser source of known parameters. Our findings would be of value to laser engineers and the community interested in nonlinear visual perception.

The aim of this paper is to present a novel method, called Adaptive Edge Detection (AED), of extraction of precise pupil edge coordinates from eye image characterized by reflections of external illuminators and laser beams. The method is used for monitoring of pupil size and position during psychophysical tests of two-photon vision performed by dedicated optical set-up. Two-photon vision is a new phenomenon of perception of short-pulsed near infrared laser beams focused on human retina as having color of half of their wavelength and it is caused by two-photon absorption occurring in human photoreceptors during safe illumination conditions. The AED method is constructed of four basic image processing operations of computational complexity of order approx. O(width*height), which makes possible to use it in real time applications. Furthermore, to achieve high resistance of AED method for presence of light reflections on images as well as other difficulties, we apply machine learning model - Haar feature based cascade (Hfbc) classifier. After implementation of trained Hfbc classifier, our method is able to find pupil edge on images, for which it was failed with this before. Finally, we obtain pupil radius and central pixel coordinates even for images containing eyelashes, changed position of illuminator or presence of reflections caused by stimulating laser beam, which significantly improves pupil edge detection efficiency.