Two-photon vision - Seeing colors in infrared.

This review discusses the current state of knowledge regarding the phenomenon called two-photon vision. It involves the visual perception of pulsed infrared beams in the range of 850-1200 nm as having colors corresponding to one-half of the IR wavelengths. It is caused by two-photon absorption (TPA), which occurs when the visual photopigment interacts simultaneously with two infrared photons. The physical mechanism of TPA is described, and implications about the efficiency of the process are considered. The spectral range of two-photon vision is defined, along with a detailed discussion of the known differences in color perception between normal and two-photon vision. The quadratic dependence of the luminance of two-photon stimuli on the power of the stimulating beam is also explained. Examples of recording two-photon vision in the retinas of mice and monkeys are provided from the literature. Finally, applications of two-photon vision are discussed, particularly two-photon microperimetry, which has been under development for several years; and the potential advantages of two-photon retinal displays are explained.

Laser pulse train parameters determine the brightness of a two-photon stimulus.

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.

In vivo imaging of the human retina using a two-photon excited fluorescence ophthalmoscope.

Noninvasive imaging of endogenous retinal fluorophores, including vitamin A derivatives, is vital to developing new treatments for retinal diseases. Here, we present a protocol for obtaining in vivo two-photon excited fluorescence images of the fundus in the human eye. We describe steps for laser characterization, system alignment, positioning human subjects, and data registration. We detail data processing and demonstrate analysis with example datasets. This technique allays safety concerns by allowing for the acquisition of informative images at low laser exposure. For complete details on the use and execution of this protocol, please refer to Boguslawski et al. (2022).1.

Femtosecond Er-doped fiber laser source tunable from 872 to 1075 nm for two-photon vision studies in humans.

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.

Two-Photon Vision in Age-Related Macular Degeneration: A Translational Study.

The recently introduced term "two-photon vision" relates to the visual perception resulting from a simultaneous absorption of two photons by photoreceptors. In this study, we determined two-photon retinal sensitivity in age-related macular degeneration (AMD) and compared it that in normal aging. Microperimetry was performed with visible (white) light and infrared (IR) light, which was perceived as green in the two-photon stimulation. In total, 45 subjects were included with one (better) eye studied. Furthermore, best-corrected visual acuity (VA) and ocular straylight were assessed. AMD resulted in decreased median (interquartile range) logMAR VA, i.e., 0.15 (0.05; 0.24), which in normal eyes was −0.02 (−0.06; 0.02). The two groups showed comparable straylight levels. Sensitivity to IR light was significantly lower in the AMD group (p < 0.001): 8.3 (7.4, 9.3) dB than in controls 10.7 (9.7, 11.2) dB. AMD also significantly affected visible light sensitivity (p < 0.001): 14.0 (11.0; 15.5) dB vs. 18.0 (16.3; 18.9) dB. Notably, the two-photon approach yielded a lower data spread. In conclusion, AMD considerably impairs retinal sensitivity measured in the single- and two-photon realm. However, two-photon-vision microperimetry may improve the testing accuracy and offer an additional diagnostic parameter (beyond VA measurements) for retinal function assessment.

Infrared- and white-light retinal sensitivity in glaucomatous neuropathy.

Glaucoma causes irreversible neuropathy, which, untreated, may lead to blindness. In this case-control study, we measured two-photon infrared (IR) light sensitivity in glaucomatous eyes to propose a new method to quantify the visual loss. In total, 64 patients were recruited with an equal distribution between glaucoma and control groups. Retinal sensitivity to IR light was assessed using a two-photon excitation device. A fundus-driven microperimeter was used to measure retinal sensitivity to visible light. The retinal nerve fiber layer (RNFL) thickness was quantified automatically with optical coherence tomography. The IR sensitivity of glaucoma and control eyes differed significantly (P = .003): 9.8 (6.5 to 13.1) dB vs. 10.9 (8.2 to 13.0) dB. Although in the visible-light microperimetry, retinal sensitivity was decreased in glaucoma (17.0, range: 6.9 to 20.0 dB) compared to the controls (17.7, range: 11.6 to 20.0 dB), this difference did not reach the significance level. A significant thinning of the RNFL in the glaucoma group was observed (P < .001). IR sensitivity significantly correlated with the RNFL in three of the four assessed quadrants instead of only one in visible-light microperimetry. Although further research is needed, this proof-of-concept study suggests that IR-light sensitivity can be used to support the detection of glaucomatous neuropathy.

In vivo imaging of the human eye using a 2-photon-excited fluorescence scanning laser ophthalmoscope.

BackgroundNoninvasive 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.MethodsWe 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.ResultsWe 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.ConclusionOur 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.FundingNIH, 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.

Pupillary Light Reflex Induced by Two-Photon Vision.

Purpose: Two-photon vision relies on the perception of pulsed infrared light due to two-photon absorption in visual pigments. This study aimed to measure human pupil reaction caused by a two-photon 1040-nm stimulus and compare it with pupil responses elicited by 520-nm stimuli of similar color. Methods: Pupillary light reflex (PLR) was induced on 14 dark-adapted healthy subjects. Three types of fovea-centered stimuli of 3.5° diameter were tested: spirals formed by fast scanning 1040-nm (infrared [IR] laser) or 520-nm (visible [VIS] laser) laser beams and uniformly filled circle created by 520-nm LED (VIS light-emitting diode [LED]). The power of visible stimuli was determined with a dedicated procedure to obtain the same perceived brightness equivalent as for 800 µW used for two-photon stimulation. Results: The minimum pupil diameter for IR laser was 88% ± 10% of baseline, significantly larger than for both VIS stimuli: 74% ± 10% (laser) and 69% ± 9% (LED). Mean constriction velocity and time to maximum constriction had significantly smaller values for IR than for both VIS stimuli. Latency times were similar for IR and VIS lasers and slightly smaller for VIS LED. Conclusions: The two-photon stimulus caused a considerably weaker pupil reaction than one-photon stimuli of the same shape, brightness, and similar color. The smaller pupil response may be due to weaker two-photon stimulation of rods relative to cones as previously observed for two-photon vision. Contrary to normal vision, in a two-photon process the stray light is not perceived, which might reduce the number of stimulated photoreceptors and further weaken the PLR.

Two-photon microperimetry with picosecond pulses.

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 (λc = 1028.4 nm, τp = 12.2 ps, Frep = 19.17 MHz) and a solid-state laser (λc = 1043.3 nm, τp = 0.253 ps, Frep = 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.

Clinical Application of Infrared-Light Microperimetry in the Assessment of Scotopic-Eye Sensitivity.

Purpose: 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. Translational Relevance: IR-light microperimetry could be applied clinically to measure visual function.

Two-photon microperimetry: sensitivity of human photoreceptors to infrared light.

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.

Sensitivity of Mammalian Cone Photoreceptors to Infrared Light.

Two-photon vision arises from the perception of pulsed infrared (IR) laser light as color corresponding to approximately half of the laser wavelength. The physical process responsible for two-photon vision in rods has been delineated and verified experimentally only recently. Here, we sought to determine whether IR light can also be perceived by mammalian cone photoreceptors via a similar activation mechanism. To investigate selectively mammalian cone signaling in mice, we used animals with disabled rod signal transduction. We found that, contrary to the expected progressive sensitivity decrease based on the one-photon cone visual pigment spectral template, the sensitivity of mouse cone photoreceptors decreases only up to 800 nm and then increases at 900 nm and 1000 nm. Similarly, in experiments with the parafoveal region of macaque retinas, we found that the spectral sensitivity of primate cones diverged above the predicted one-photon spectral sensitivity template beyond 800 nm. In both cases, efficient detection of IR light was dependent on minimizing the dispersion of the ultrashort light pulses, indicating a non-linear two-photon activation process. Together, our studies demonstrate that mammalian cones can be activated by near IR light by a nonlinear two-photon excitation. Our results pave the way for the creation of a two-photon IR-based ophthalmoscope for the simultaneous imaging and functional testing of human retinas as a novel tool for the diagnosis and treatment of a wide range of visual disorders.

Periscope for noninvasive two-photon imaging of murine retina in vivo.

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.

Human infrared vision is triggered by two-photon chromophore isomerization.

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 two-photon isomerization of visual pigments.

Multimodal instrument for high-sensitivity autofluorescence and spectral optical coherence tomography of the human eye fundus.

In this paper we present a multimodal device for imaging fundus of human eye in vivo which combines functionality of autofluorescence by confocal SLO with Fourier domain OCT. Native fluorescence of human fundus was excited by modulated laser beam (λ = 473 nm, 20 MHz) and lock-in detection was applied resulting in improving sensitivity. The setup allows for acquisition of high resolution OCT and high contrast AF images using fluorescence excitation power of 50-65 µW without averaging consecutive images. Successful functioning of constructed device have been demonstrated for 8 healthy volunteers of different age ranging from 24 to 83 years old.

Fish products available in Polish market--assessment of the nutritive value and human exposure to dioxins and other contaminants.

Chemical analyses were performed on one hundred and twenty of the most popular varieties of fish products (smoked fish, salted fish, and marinated fish) of the fish market in Poland. The contents of the nutritive substances of fish products (protein, micro- and macronutrients, vitamins A(1), D(3), E, and fatty acids) and the chosen contaminant (toxic metals--mercury, cadmium, lead, arsenic; dioxin/furans--PCDDFs; dioxin-like PCB--dl-PCBs; seven congeners of polybrominated diphenyl ethers--PBDEs; organochlorine pesticides--SigmaDDT, HCB, SigmaHCH and marker polychlorinated biphenyls--PCB(7)) levels were determined. It was confirmed that fish products are a good source of digestible proteins, iodine, selenium, and vitamin D(3). The fundamental nutritive benefit of processed fish lies in its highly beneficial fatty acid composition, which is what imparts them healthy nutritive qualities. The high content of long-chain polyunsaturated fatty acids (LC-PUFAs), which is not noted in other food products, is particularly important. The majority of contaminants studied were present in low levels. The possible threats, particularly in the case of pregnant/nursing women and young children, can pose the levels of dioxin/furans (PCDD/Fs) and dioxin-like (dl-PCBs) in smoked Baltic salmon and smoked sprat, elevated in a relation to particular requirements concerning the content of sum of PCDD/Fs and dl-PCBs in fish (8pg WHO-TEQg(-1)). The health benefits and risks stemming from consumption of fish products were determined according to the Provisional Tolerable Weekly Intake (PTWI) for chosen contaminants (Cd, Hg, As, PCDD/Fs+dl-PCB) and the quantity of ingredients that render a fish diet healthy based on data from the EFSA Journal [EFSA (European Food Safety Authority), 2005. Opinion of the scientific panel on contaminants in the food chain on a request from the European Parliament related to the safety assessment of wild and farmed fish. EFSA J. 236, 1-118]. In regard of high content of LC-PUFAa and other nutritive ingredients, fish products available in Polish market may be considered as healthy food. However, many authors point at contaminants (methylmercury, PCDD/Fs) occurring in fish and fish products as on potential health problem, and emphasize that the amount of that hazardous substances should be limited in human diet.