Vision relies on photoactivation of visual pigments in rod and cone photoreceptor cells of the retina. The human eye structure and the absorption spectra of pigments limit our visual perception of light. Our visual perception is most responsive to stimulating light in the 400-to 720-nm (visible) range. First, we demonstrate by psychophysical experiments that humans can perceive infrared laser emission as visible light. Moreover, we show that mammalian photoreceptors can be directly activated by near infrared light with a sensitivity that paradoxically increases at wavelengths above 900 nm, and display quadratic dependence on laser power, indicating a nonlinear optical process. Biochemical experiments with rhodopsin, cone visual pigments, and a chromophore model compound 11-cis-retinyl-propylamine Schiff base demonstrate the direct isomerization of visual chromophore by a two-photon chromophore isomerization. Indeed, quantum mechanics modeling indicates the feasibility of this mechanism. Together, these findings clearly show that human visual perception of near infrared light occurs by twophoton isomerization of visual pigments.visual pigment | two-photon absorption | rhodopsin | transretinal electrophysiology | multiscale modeling H uman vision is generally believed to be restricted to a visible light range, although >50% of the sun's radiation energy that reaches earth is in the infrared (IR) range (1). Human rod and cone visual pigments with the 11-cis-retinylidene chromophore absorb in the visible range, with absorption monotonically declining from their maxima of 430-560 nm toward longer wavelengths. The spectral sensitivity of human dim light perception matches well with the absorption spectrum of the rod visual pigment, rhodopsin (2, 3). Activation of visual pigments is temperature independent around their absorption peaks (λ max ), but at longer wavelengths, the lower energy photons must be supplemented by heat to achieve chromophore photoisomerization (4). Long wavelength-sensitive visual pigments of vertebrates exhibit maximal absorption at the ∼500-to ∼625-nm range. Pigments with λ max > 700 nm are theoretically possible, but the high noise due to spontaneous thermal activation would render them impractical (5). At human body temperature and with 1,050-nm stimulation, the sensitivity of the peripheral retina to one-photon (1PO) stimulation is less than 10 −12 of its maximum value at 505 nm (4, 6). Indeed, reports about human IR vision can be found in the literature, although they are fragmentary and do not describe the mechanism of this phenomenon.With the invention of radar during World War II, it was immediately questioned if pilots could detect high intensity radiation in the IR range of the spectrum. Wald and colleagues reported that at wavelengths above 800 nm, rod photoreceptors become more sensitive than cones, resulting in perception of IR signals as white light selectively in the peripheral retina (6). They proposed that relative spectral sensitivity declines monotonically toward longer wavele...

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Microperimetry is a subjective ophthalmologic test used to assess retinal function at various specific and focal locations of the visual field. Historically, visible light has been described as ranging from 400 to 720 nm. However, we previously demonstrated that infra-red light can initiate visual transduction in rod photoreceptors by a mechanism of two-photon absorption by visual pigments. Here we introduce a newly designed and constructed two-photon microperimeter. We provide for the first time evidence of the presence of a nonlinear process occurring in the human retina based on psychophysical tests using newly developed instrumentation. Since infra-red light penetrates the aged front of the eye better than visible light, it has the potential for improved functional diagnostics in patients with age-related visual disorders.

Two-photon microscopy allows visualization of subcellular structures in the living animal retina. In previously reported experiments it was necessary to apply a contact lens to each subject. Extending this technology to larger animals would require fitting a custom contact lens to each animal and cumbersome placement of the living animal head on microscope stage. Here we demonstrate a new device, periscope, for coupling light energy into mouse eye and capturing emitted fluorescence. Using this periscope we obtained images of the RPE and their subcellular organelles, retinosomes, with larger field of view than previously reported. This periscope provides an interface with a commercial microscope, does not require contact lens and its design could be modified to image retina in larger animals.

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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 two-photon excited fluorescence scanning laser ophthalmoscope and spectrally resolved images of the human retina based on two-photon excitation (TPE) with near-infrared (IR) light. A custom Er:fiber laser with integrated pulse selection, along with intelligent post-processing 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.

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The eye can see pulsed near-infrared (IR) radiation with the color corresponding to half of the wavelength used. Until recently, the technology required for measuring IR vision was confined to optical laboratories and was not studied clinically. The current investigation sought to determine the values for IR thresholds in a healthy population. Methods: IR-light threshold was measured in 45 healthy participants, aged from 21 to 70 years. Ten patients with retinal pathology were included for comparison. Ocular media clarity was assessed with a straylight parameter. The sensitivity of dark-adapted eyes (expressed on a 0-26 dB scale) were tested using an IR microperimeter. The device consists of a femtosecond laser that emits 1045 nm light to project a stimulus at the retina. Results: All participants were able to see the IR stimulus, which they perceived as green, and all performed the test. Measurements at seven locations revealed lower sensitivity at the fovea (15.5 dB) than in paracentral regions (18.2 dB). We noted a significant straylight increase with age. Although, in our study population, it was only a slight,-0.18 dB decline per decade of the average IR-sensitivity. The retinal-pathology group demonstrated impaired sensitivity to IR light. Conclusions: We showed that IR-light sensitivity does not significantly decrease with age despite a straylight increase. A reference level for the IR threshold was proposed. The application of IR-light microperimetry can be extended to the assessment of retinal pathology.