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...
High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome these hurdles, we improved TPE efficiency by controlling temporal properties of the excitation light and employed phasor analyses to FLIM and spectral data in mouse models of retinal diseases. Modeling of retinal photodamage revealed that plasma-mediated effects do not play a role and that melanin-related thermal effects are mitigated by reducing pulse repetition frequency. By using noninvasive TPE imaging we identified molecular components of individual granules in the retinal pigment epithelium and present their analytical characteristics.
Corneal evaluation in ophthalmology necessitates cellular-resolution and fast imaging techniques allowing accurate diagnoses. Currently, the fastest volumetric imaging technique is Fourier-domain full-field optical coherence tomography (FD-FF-OCT) that uses a fast camera and a rapidly tunable laser source. Here, we demonstrate high-resolution, highspeed, non-contact corneal volumetric imaging in vivo with FD-FF-OCT that can acquire a single 3D volume with a voxel rate of 7.8 GHz. The spatial coherence of the laser source was suppressed to prevent it from focusing to a spot on the retina, and therefore, exceeding the maximum permissible exposure (MPE). Inherently volumetric nature of FD-FF-OCT data enabled flattening of curved corneal layers. Acquired FD-FF-OCT images revealed corneal cellular structures, such as epithelium, stroma and endothelium, as well as subbasal and midstromal nerves.
Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms of retinal diseases and for developing therapies for their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate in metabolic processes of the retina is highly attenuated by the anterior segment of the human eye. In contrast, 2-photon excitation fluorescence imaging with pulsed infrared light overcomes this obstacle. Reducing retinal exposure to laser radiation remains a major barrier in advancing this technology to studies in humans. To increase fluorescence intensity and reduce the requisite laser power, we modulated ultrashort laser pulses with high-order dispersion compensation and applied sensorless adaptive optics and custom image recovery software and observed an over 300% increase in fluorescence of endogenous retinal fluorophores when laser pulses were shortened from 75 fs to 20 fs. No functional or structural changes to the retina were detected after exposure to 2-photon excitation imaging light with 20-fs pulses. Moreover, wide bandwidth associated with short pulses enables excitation of multiple fluorophores with different absorption spectra and thus can provide information about their relative changes and intracellular distribution. These data constitute a substantial advancement for safe 2-photon fluorescence imaging of the human eye.
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.
Two-photon fluorescence microscopy (TPM) is now being used routinely to image live cells for extended periods deep within tissues, including the retina and other structures within the eye . However, very low laser power is a requirement to obtain TPM images of the retina safely. Unfortunately, a reduction in laser power also reduces the signal-to-noise ratio of collected images, making it difficult to visualize structural details. Here, image registration and averaging methods applied to TPM images of the eye in living animals (without the need for auxiliary hardware) demonstrate the structural information obtained with laser power down to 1 mW. Image registration provided between 1.4% and 13.0% improvement in image quality compared to averaging images without registrations when using a high-fluorescence template, and between 0.2% and 12.0% when employing the average of collected images as the template. Also, a diminishing return on image quality when more images were used to obtain the averaged image is shown. This work provides a foundation for obtaining informative TPM images with laser powers of 1 mW, compared to previous levels for imaging mice ranging between 6.3 mW [Palczewska G., Nat Med.20, 785 (2014) Sharma R., Biomed. Opt. Express4, 1285 (2013)].
Optical Coherence Imaging (OCI) including Optical Coherence Tomography (OCT) and Optical Coherence Microscopy (OCM) uses interferometric detection to generate high-resolution volumetric images of the sample at high speeds. Such capabilities are significant for in vivo imaging, including ophthalmology, brain, intravascular imaging, as well as endoscopic examination. Instrumentation and software development allowed to create many clinical instruments. Nevertheless, most of OCI setups scan the incident light laterally. Hence, OCI can be further extended by wide-field illumination and detection. This approach, however, is very susceptible to the so-called crosstalk-generated noise. Here, we describe our novel approach to overcome this issue with spatio-temporal optical coherence manipulation (STOC), which employs spatial phase modulation of the incident light. Full Text: PDF ReferencesL. Wang, P. P. Ho, C. Liu, G. Zhang, and R. R. Alfano, "Ballistic 2-D Imaging Through Scattering Walls Using an Ultrafast Optical Kerr Gate", Science 253, 769-771 (1991). CrossRef D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson, W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and et al., "Optical coherence tomography", Science 254, 1178-1181 (1991). CrossRef J. A. Izatt, E. A. Swanson, J. G. Fujimoto, M. R. Hee, and G. M. Owen, "Optical coherence microscopy in scattering media", Opt. Lett. 19, 590-592 (1994). CrossRef D. Borycki, M. Nowakowski, and M. Wojtkowski, "Control of the optical field coherence by spatiotemporal light modulation", Opt. Lett. 38, 4817-4820 (2013). CrossRef D. Borycki, M. Hamkalo, M. Nowakowski, M. Szkulmowski, and M. Wojtkowski, "Spatiotemporal optical coherence (STOC) manipulation suppresses coherent cross-talk in full-field swept-source optical coherence tomography", Biomed. Opt. Express 10, 2032-2054 (2019). CrossRef P. Stremplewski, E. Auksorius, P. Wnuk, L. Kozon, P. Garstecki, and M. Wojtkowski, "In vivo volumetric imaging by crosstalk-free full-field OCT", Optica 6, 608-617 (2019). CrossRef L. Vabre, A. Dubois, and A. C. Boccara, "Thermal-light full-field optical coherence tomography", Opt. Lett. 27, 530-532 (2002). CrossRef M. Laubscher, M. Ducros, B. Karamata, T. Lasser, and R. Salathé, "Video-rate three-dimensional optical coherence tomography", Opt. Express 10, 429-435 (2002). CrossRef Dubois and A. C. Boccara, Full-Field Optical Coherence Tomography, (Springer Berlin Heidelberg, Berlin, Heidelberg, 2008), pp. 565-591. CrossRef O. Thouvenin, K. Grieve, P. Xiao, C. Apelian, and A. C. Boccara, "En face coherence microscopy [Invited]", Biomedical Opt. Express 8, 622-639 (2017). CrossRef F. Fercher, C. K. Hitzenberger, M. Sticker, E. Moreno-Barriuso, R. Leitgeb, W. Drexler, and H. Sattmann, "A thermal light source technique for optical coherence tomography", Optics Commun. 185, 57-64 (2000). CrossRef R. A. Leitgeb, "En face optical coherence tomography: a technology review [Invited]", Biomed Opt Express 10, 2177-2201 (2019). CrossRef J. Fujimoto and W. Drexler, Introduction to Optical Coherence Tomography, (Springer, Berlin, Heidelberg, 2008), pp. 1-45. CrossRef J. A. Izatt, M. A. Choma, and A.-H. Dhalla, Theory of Optical Coherence Tomography, (Springer International Publishing, Cham, 2015), pp. 65-94. CrossRef
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