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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.

Pulsed near-infrared (NIR) light sources can be successfully applied for both imaging and functional testing of the human eye, as published recently1. These two groups of applications have different requirements. For imaging applications, the most preferable is invisible scanning beam while efficiently visible stimulating beam is preferable for functional testing applications. The functional testing of human eye using NIR laser beams is possible due to two-photon vision (2PV) phenomenon. 2PV enables perception of pulsed near-infrared laser light as color corresponding to approximately half of the laser wavelength2. This study aims to characterize two-photon vision thresholds for various pulse lengths from a solid- state sub-picosecond laser (λc = 1043.3 nm, Frep = 62.65 MHz), either of 253 fs duration or elongated by Martinez- type stretcher to 2 ps, and fiber-optic picosecond laser (λc = 1028.4 nm, Frep = 19.19 MHz, p = 12.2 ps).

The two- and one-photon visual sensitivities (2phS and 1phS) of cataract patients were tested before and after IOL surgery. Mean change in 2phS was twice smaller than 1phS indicating that 2phS is less affected by cataract.

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.

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.

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

Human subjects can detect infrared light at wavelengths over 1000 nm perceived as visible of the corresponding half wavelength. This is due to a two-photon process and requires the use of pulsed light sources well focused within the retina. We have developed an experimental system to measure, for the first time, the visual resolution of the eye when is stimulated with infrared (1043 nm) and compared with visible light (543 nm). Scanner mirrors were used to project letters of different sizes onto the retina in both wavelengths. Subjects performed a visual test to determine the smallest letter size that was distinguishable for each wavelength for a range of defocus values. An additional optical path was used to record the retinal images of the spot after reflection in the retina and double-pass through the optical media. The best visual acuity was obtained at different defocus locations corresponding to the chromatic difference between green and infrared. Although, there was some individual variability, visual acuity was found to be similar both in visible and infrared. The use of two-photon infrared vision may have some potential applications for vision in those cases were the optical media is opaque to visible wavelengths while keeping some transparency in the infrared.

Light sensation relies on photoisomerization of chromophores in rod and cone photoreceptor cells. Spectral sensitivity of these photoreceptor cells in the retina is determined by the absorption spectra of their pigments which covers a range from 400 nm to above 700 nm. Regardless the mechanism leading to visual pigment isomerization, light sensation is triggered every time visual pigment molecules change their conformation. Thus, two-photon absorption (TPA) should produce the same result (visual sensation) as single photon absorption of light. This observation was positively verified and published by our group. During human psychophysics experiments, we found that humans can perceive light in the infrared (IR) range as colors that match half of the wavelength of the applied laser beam. Other experiments and theoretical research, such as mouse electrophysiology, biochemical studies of TPA in rhodopsin or molecular modeling studies, confirmed that visual sensation can be triggered by TPA. There are few publications describing human near infrared (NIR) perception and no formal proposals to use this phenomenon to improve ophthalmic diagnosis and monitor treatment. Here we report that the use of novel instrumentation revealed that the sensitivity threshold for NIR vision depends on age.

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.

Laser ablation was applied for surface cleaning and spectroscopic diagnostics of historical paper documents and model samples in the framework of the conservation projects. During cleaning the spectra of ablation products were recorded by means of the LIBS technique which allowed for nearly non-destructive identification of surface layers such as contaminants, substrate and pigments. For consecutive laser pulses a strong decrease of band intensities of the emission lines of Ca, Na, K, Al and Fe ascribed to contaminants were observed. The effect was used for monitoring of the cleaning progress of stained paper. For surface cleaning and spectra excitation the Q-switched Nd:YAG laser of 6 ns pulsewidth operating at wavelengths of 266, 355, 532, and 1064 nm and of fluence selected from the range 0.3–0.9 J/cm 2 was applied. The ablation parameters were optimized in agreement with the literature and the results were confirmed by surface studies and testing of the mechanical and chemical properties, and also by the response to the ageing process of the paper substrate. In case of the model paper irradiated in the UV range at 266 and 355 nm a visual inspection revealed local damages of the cellulose fibers accompanied by a decrease of the mechanical strength of the substrate. The effect was more pronounced after artificial ageing. The best results were obtained for samples irradiated at 532 nm and at laser fluence below the damage threshold of 0.6 J/cm 2 , which is in agreement with literature.

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.

Looking for the processes in which 2 photons are involved in human eye autofluorescence intensity, we measured excited state absorption (ESA) in lipofuscin solution. Performing pump and probe experiment, we've found ESA at 850 nm.

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.

Groups of flounder ( Platichthys flesus ) females were collected in 2011 from the Vistula River and the Duoro River estuaries and corresponding reference sites in the southern Baltic Sea and Portuguese coast of the Atlantic Ocean to measure and compare the levels and profiles of polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), and polybrominated diphenyl ethers (PBDEs). The estuaries’ sediments were also investigated. Several differences were found in the POPs between the estuaries and between the two marine regions, which were highlighted by PCA. The Vistula River estuary POPs, significantly higher than in the Douro River estuary, were dominated by DDTs followed by PCBs. PBDEs levels, indifferent between the estuaries, were relatively low. The POP levels in flounder and sediment evaluated against environmental assessment criteria (EACs) indicated that none of the measured contaminants for which EAC had been established exceeded the criterion, except for CB-118 in flounder from the Vistula River estuary.

Scattering and fluorescence images provide complementary information about the health condition of the human eye, so getting them in a single measurement, using a single device may significantly improve a quality of diagnosis as it has been already demonstrated in Spectralis (Heidelberg Eng.) OCT instrument. There is still challenge to improve quality of fundus autofluorescence (FAF) images. The biggest obstacle in obtaining in vivo images of sufficient quality is very low fluorescence signal. For eye safety reasons, and because of patient comfort, using highpower fluorescence excitation is not an adequate solution to the low signal problem. In this contribution we show a new detection method in the retinal autofluorescence imaging, which may improve the sensitivity. We used a fast modulated (up to 500 MHz) diode laser of wavelength 473 nm and detected fluorescence in the spectral range 500-680 nm by photomultiplier and lock-in amplifier. Average power of the collimated blue beam on the cornea used for FAF measurements was set to 50 μW, 10 μW, and even 4.5 μW.

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.

Techniques of laser emission spectroscopy such as LIPS and LIF are applied to identify pastels and pigments composition for the use in conservation of historical documents. The question of data reliable for pigment identification by these techniques is considered. For model samples made of the cotton paper of chemical composition corresponding to the historical ones, and coated with pastels of different colors the LIPS and LIF spectra are recorded. Samples are excited by the pulsed Nd:YAG laser operating at 355 or 266 nm. The reference Raman spectra are collected, too. Bands characteristic for the blue pigments: PB15, PB29; violet one PV16, and yellow PY 184 are identified exclusively by LIPS in agreement with literature. Also additives such as the barium white found in the Scarlet pastel, and ultramarine (Na 8-10 [Al 6 Si 6 O 24 ]S 2-4 ) with titanium white (TiO 2 ) in the Phthalo Blue are identified, and confirmed by the Raman technique as well. The pigments anthraquinone (PR 168), isoindolinone (PY 110) and monoazo (PY 74) are not revealed. In the LIF spectra only a broad band centered at 612 nm and corresponding to anthraquinone (red pastel) can be clearly assigned.

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.