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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.

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 fluorescence scanning laser ophthalmoscope (TPEF-SLO) 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. 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.

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 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.

The consumption of raw or inadequately cooked marine fish can lead to several disorders caused by the ingestion of viable anisakid nematodes. Although anisakid larvae can be killed by subzero temperatures, making freezing an important control measure for this potential health hazard, these parasites can survive freezing under some conditions. Therefore, the aim of the present study was to experimentally evaluate the time-temperature conditions needed to kill Anisakis simplex and Pseudoterranova spp. The effectiveness of freezing was tested on two species of fish: cod, Gadus morhua from the North Atlantic, and herring, Clupea harengus membras from the southern Baltic Sea. Samples, which comprised skinless fillets of cod (n = 40) with visible parasites and whole herring (n = 240), were separately frozen at − 15, − 18, or − 20 °C for 24 h, or at − 20 °C for 48 h in the single-compressor freezer and at − 20, − 25, or − 35 °C for 24 h in the double-compressor freezer. After thawing, parasites were stained with malachite green and examined under the microscope for viability. All A. simplex and Pseudoterranova spp. larvae in cod fillets died at a temperature of − 15 °C or lower. However, freezing did not kill all the A. simplex larvae in whole herring: spontaneous movement of these parasites was observed in samples stored in the single-compressor freezer at − 15, − 18, and − 20 °C over 24 h. Our results demonstrate that the freezing procedure must consider both the capability of the freezing device and the nature of the fish product to ensure consumer safety.

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.

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 absorption occurring in photoreceptors of human eye is responsible for perception of pulsed infrared laser beams. Color of perceived light corresponds to about half of wavelength of stimulating beam. This recently discovered phenomenon is called two-photon vision. However it is yet not clear how such phenomena could impact an ophthalmic diagnosis and therefore further studies are required. In this paper we present the system that enables future psychophysical experiments to extend knowledge about two-photon vision and its diagnostic value by identifying differences between two-photon and single-photon vision. Full Text: PDF References D. A. Atchison, G. Smith, Optics of the human eye (Butterworth-Heinemann 2000) CrossRef G. Palczewska et al. "Human infrared vision is triggered by two-photon chromophore isomerization", Proceedings of the National Academy of Sciences 111, 1428 (2014). CrossRef P. Artal, S. Manzanera, K. Komar, A. Gambin-Regadera, M. Wojtkowski, "Visual acuity in two-photon infrared vision", Optica 4, 1488 (2017). CrossRef K. Komar et al. "Posterior Eye Shape Measurement With Retinal OCT Compared to MRI", Investigative Ophthalmology & Visual Science 57, 196 (2016). CrossRef K. Komar et al. "Two-photon visual sensitivity of human cones - a psychophysical study", Investigative Ophthalmology & Visual Science 59, 4049 (2018). DirectLink S. A. Burns,R.H. Webb, Handbook of optics 28 (New York, McGraw Hill 1994) DirectLink R. W. Nygaard, T. E. Frumkes, "Calibration of the retinal illuminance provided by maxwellian views", Vision Research (1982) CrossRef P. Melillo et al. "Pupillometric analysis for assessment of gene therapy in Leber Congenital Amaurosis patients", Biomedical engineering online 11, 40 (2012) CrossRef P. A. Stanley, A. K. Davies, "The effect of field of view size on steady-state pupil diameter", Ophthalmic and Physiological Optics, 15, 601 (1995) CrossRef S. Mathot, "Pupil mimicry is the result of brightness perception of the iris and pupil" Journal of Cognition 1 (2018) CrossRef M. M. Thomas, T. D. Lamb, "Light adaptation and dark adaptation of human rod photoreceptors measured from the a‐wave of the electroretinogram", The Journal of physiology 518, 479 (1999) CrossRef

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.

Humans can detect infrared light at wavelengths over 1000 nm, perceived as visible light of the corresponding half wavelength. This is due to a two-photon (2P) absorption process, which requires sufficiently large amounts of luminous energy. For safety reasons, this energy must be delivered by pulsed light sources well focused in the retina. Although this effect has been known for several decades, the spatial properties of 2P vision in comparison to normal vision have not yet elucidated. We have developed a new experimental system to measure, for the first time, to the best of our knowledge, visual acuity mediated by 2P absorption and compare it against that with visible light. The spatial resolution of 2P infrared vision is the same as in normal visible light. However, the use of 2P infrared vision may have some future potential applications, for example, in permitting vision in those cases with opaque optical media to visible wavelengths while keeping some transparency in the infrared.

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.

Osmium and ruthenium can be selectively isolated from a mixture of the noble metals by distillation in the form of OsO4 and RuO4. In hydrochloric (or sulfuric) acid media and in the presence of iodide, OsO4 is selectively reduced to osmium(IV) and the OsI62- complex is formed. Quantitative conversion of Os(VIII) into this complex takes place in 0.8M I- solutions and in 0.04M HCl (or 0.1M H2SO4) within 5 (or 10min), respectively. The OsI62- complex reacts with trioctylamine ((C8H17)3N) and the ion associate ((C8H17)3NH+)2OsI62- is formed. The ion associate can be isolated from aqueous solutions by extraction into carbon tetrachloride. Both the OsI62- complex and the ion pair with trioctylamine can be used for spectrophotometric determination of osmium directly in aqueous solutions or after extraction into the organic solvent. Ruthenium does not interfere with the determination of osmium in the form of the OsI62- complex.

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.

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.