Distinct mouse models of Stargardt disease display differences in pharmacological targeting of ceramides and inflammatory responses.

Mutations in many visual cycle enzymes in photoreceptors and retinal pigment epithelium (RPE) cells can lead to the chronic accumulation of toxic retinoid byproducts, which poison photoreceptors and the underlying RPE if left unchecked. Without a functional ATP-binding cassette, sub-family A, member 4 (ABCA4), there is an elevation of all-trans-retinal and prolonged buildup of all-trans-retinal adducts, resulting in a retinal degenerative disease known as Stargardt-1 disease. Even in this monogenic disorder, there is significant heterogeneity in the time to onset of symptoms among patients. Using a combination of molecular techniques, we studied Abca4 knockout (simulating human noncoding disease variants) and Abca4 knock-in mice (simulating human misfolded, catalytically inactive protein variants), which serve as models for Stargardt-1 disease. We compared the two strains to ascertain whether they exhibit differential responses to agents that affect cytokine signaling and/or ceramide metabolism, as alterations in either of these pathways can exacerbate retinal degenerative phenotypes. We found different degrees of responsiveness to maraviroc, a known immunomodulatory CCR5 antagonist, and to the ceramide-lowering agent AdipoRon, an agonist of the ADIPOR1 and ADIPOR2 receptors. The two strains also display different degrees of transcriptional deviation from matched WT controls. Our phenotypic comparison of the two distinct Abca4 mutant-mouse models sheds light on potential therapeutic avenues previously unexplored in the treatment of Stargardt disease and provides a surrogate assay for assessing the effectiveness for genome editing.

Stress resilience-enhancing drugs preserve tissue structure and function in degenerating retina via phosphodiesterase inhibition.

Chronic, progressive retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa, arise from genetic and environmental perturbations of cellular and tissue homeostasis. These disruptions accumulate with repeated exposures to stress over time, leading to progressive visual impairment and, in many cases, legal blindness. Despite decades of research, therapeutic options for the millions of patients suffering from these disorders remain severely limited, especially for treating earlier stages of pathogenesis when the opportunity to preserve the retinal structure and visual function is greatest. To address this urgent, unmet medical need, we employed a systems pharmacology platform for therapeutic development. Through integrative single-cell transcriptomics, proteomics, and phosphoproteomics, we identified universal molecular mechanisms across distinct models of age-related and inherited retinal degenerations, characterized by impaired physiological resilience to stress. Here, we report that selective, targeted pharmacological inhibition of cyclic nucleotide phosphodiesterases (PDEs), which serve as critical regulatory nodes that modulate intracellular second messenger signaling pathways, stabilized the transcriptome, proteome, and phosphoproteome through downstream activation of protective mechanisms coupled with synergistic inhibition of degenerative processes. This therapeutic intervention enhanced resilience to acute and chronic forms of stress in the degenerating retina, thus preserving tissue structure and function across various models of age-related and inherited retinal disease. Taken together, these findings exemplify a systems pharmacology approach to drug discovery and development, revealing a new class of therapeutics with potential clinical utility in the treatment or prevention of the most common causes of blindness.

Noninvasive two-photon optical biopsy of retinal fluorophores.

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.

THE LOSS OF INFRARED LIGHT SENSITIVITY OF PHOTORECEPTOR CELLS MEASURED WITH TWO-PHOTON EXCITATION AS AN INDICATOR OF DIABETIC RETINOPATHY: A Pilot Study.

PURPOSE: Human photoreceptors are sensitive to infrared light (IR). This sensitivity can be used as a novel indicator of retinal function. Diabetic retinopathy patients were assessed using in vivo two-photon excitation and compared their scotopic IR threshold with that of healthy patients. METHODS: Sixty-two participants, 28 healthy and 34 with diabetic retinopathy, underwent a comprehensive eye examination, where visual acuity and contrast sensitivity were assessed. Infrared thresholds were measured in the fovea and parafovea following 30-minute dark adaptation. A two-photon excitation device was used with integrated pulsed laser light (1,045 nm) for sensitivity testing and scanning laser ophthalmoscopy for fundus imaging. RESULTS: The mean Snellen visual acuity of diabetic patients (6/7.7) was worse than that of the healthy patients (6/5.5), which was significantly different (P < 0.001). Disease patients had decreased contrast sensitivity, especially at 6 and 18 cycles/degree. The mean retinal sensitivity to IR light in eyes with diabetic retinopathy (11.6 ± 2.0 dB) was significantly (P < 0.001) lower than that in normal eyes (15.5 ± 1.3 dB). CONCLUSION: Compared with healthy control subjects, the IR light sensitivity of diabetic patients was significantly impaired. Two-photon measurements can be used in the assessment of retinal disease, but further studies are needed to validate IR light stimulation in various stages of diabetic retinopathy.

Photic generation of 11-cis-retinal in bovine retinal pigment epithelium.

Photoisomerization of the 11-cis-retinal chromophore of rod and cone visual pigments to an all-trans-configuration is the initiating event for vision in vertebrates. The regeneration of 11-cis-retinal, necessary for sustained visual function, is an endergonic process normally conducted by specialized enzyme systems. However, 11-cis-retinal also can be formed through reverse photoisomerization from all-trans-retinal. A nonvisual opsin known as retinal pigment epithelium (RPE)-retinal G-protein-coupled receptor (RGR) was previously shown to mediate visual chromophore regeneration in photic conditions, but conflicting results have cast doubt on its role as a photoisomerase. Here, we describe high-level production of 11-cis-retinal from RPE membranes stimulated by illumination at a narrow band of wavelengths. This activity was associated with RGR and enhanced by cellular retinaldehyde-binding protein (CRALBP), which binds the 11-cis-retinal produced by RGR and prevents its re-isomerization to all-trans-retinal. The activity was recapitulated with cells heterologously expressing RGR and with purified recombinant RGR. Using an RGR variant, K255A, we confirmed that a Schiff base linkage at Lys-255 is critical for substrate binding and isomerization. Single-cell RNA-Seq analysis of the retina and RPE tissue confirmed that RGR is expressed in human and bovine RPE and Müller glia, whereas mouse RGR is expressed in RPE but not in Müller glia. These results provide key insights into the mechanisms of physiological retinoid photoisomerization and suggest a novel mechanism by which RGR, in concert with CRALBP, regenerates the visual chromophore in the RPE under sustained light conditions.

Protective Effect of a Locked Retinal Chromophore Analog against Light-Induced Retinal Degeneration.

Continuous regeneration of the 11-cis-retinal visual chromophore from all-trans-retinal is critical for vision. Insufficiency of 11-cis-retinal arising from the dysfunction of key proteins involved in its regeneration can impair retinal health, ultimately leading to loss of human sight. Delayed recovery of visual sensitivity and night blindness caused by inadequate regeneration of the visual pigment rhodopsin are typical early signs of this condition. Excessive concentrations of unliganded, constitutively active opsin and increased levels of all-trans-retinal and its byproducts in photoreceptors also accelerate retinal degeneration after light exposure. Exogenous 9-cis-retinal iso-chromophore can reduce the toxicity of ligand-free opsin but fails to prevent the buildup of retinoid photoproducts when their clearance is defective in human retinopathies, such as Stargardt disease or age-related macular degeneration. Here we evaluated the effect of a locked chromophore analog, 11-cis-6-membered ring-retinal against bright light-induced retinal degeneration in Abca4-/-Rdh8-/- mice. Using in vivo imaging techniques, optical coherence tomography, scanning laser ophthalmoscopy, and two-photon microscopy, along with in vitro histologic analysis of retinal morphology, we found that treatment with 11-cis-6-membered ring-retinal before light stimulation prevented rod and cone photoreceptor degradation and preserved functional acuity in these mice. Moreover, additive accumulation of 11-cis-6-membered ring-retinal measured in the eyes of these mice by quantitative liquid chromatography-mass spectrometry indicated stable binding of this retinoid to opsin. Together, these results suggest that eliminating excess of unliganded opsin can prevent light-induced retinal degeneration in Abca4-/-Rdh8-/- mice.

Receptor MER Tyrosine Kinase Proto-oncogene (MERTK) Is Not Required for Transfer of Bis-retinoids to the Retinal Pigmented Epithelium.

Accumulation of bis-retinoids in the retinal pigmented epithelium (RPE) is a hallmark of aging and retinal disorders such as Stargardt disease and age-related macular degeneration. These aberrant fluorescent condensation products, including di-retinoid-pyridinium-ethanolamine (A2E), are thought to be transferred to RPE cells primarily through phagocytosis of the photoreceptor outer segments. However, we observed by two-photon microscopy that mouse retinas incapable of phagocytosis due to a deficiency of the c-Mer proto-oncogene tyrosine kinase (Mertk) nonetheless contained fluorescent retinoid condensation material in their RPE. Primary RPE cells from Mertk-/- mice also accumulated fluorescent products in vitro Finally, quantification of A2E demonstrated the acquisition of retinal condensation products in Mertk-/- mouse RPE prior to retinal degeneration. In these mice, we identified activated microglial cells that likely were recruited to transport A2E-like condensation products to the RPE and dispose of the dying photoreceptor cells. These observations demonstrate a novel transport mechanism between photoreceptor cells and RPE that does not involve canonical Mertk-dependent phagocytosis.

Protein misfolding and the pathogenesis of ABCA4-associated retinal degenerations.

Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4, even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all-trans-retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4(PV/PV) knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo. Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4(-/-) and Abca4(PV/PV) mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4(-/-) mice, Abca4(PV/PV) mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients.

Two-photon microscopy reveals early rod photoreceptor cell damage in light-exposed mutant mice.

Atrophic age-related and juvenile macular degeneration are especially devastating due to lack of an effective cure. Two retinal cell types, photoreceptor cells and the adjacent retinal pigmented epithelium (RPE), reportedly display the earliest pathological changes. Abca4(-/-)Rdh8(-/-) mice, which mimic many features of human retinal degeneration, allowed us to determine the sequence of light-induced events leading to retinal degeneration. Using two-photon microscopy with 3D reconstruction methodology, we observed an initial strong retinoid-derived fluorescence and expansion of Abca4(-/-)Rdh8(-/-) mouse rod cell outer segments accompanied by macrophage infiltration after brief exposure of the retina to bright light. Additionally, light-dependent fluorescent compounds produced in rod outer segments were not transferred to the RPE of mice genetically defective in RPE phagocytosis. Collectively, these findings suggest that for light-induced retinopathies in mice, rod photoreceptors are the primary site of toxic retinoid accumulation and degeneration, followed by secondary changes in the RPE.

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.

Retinal pigmented epithelial cells obtained from human induced pluripotent stem cells possess functional visual cycle enzymes in vitro and in vivo.

Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat(-/-) and Rpe65(-/-) mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat(-/-) and Rpe65(-/-) mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases.

Primary amines protect against retinal degeneration in mouse models of retinopathies.

Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore 11-cis-retinal and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomerized product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing Food and Drug Administration (FDA)-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by MS. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that shows features of Stargardt's disease and age-related retinal degeneration.

Ultrasound characterization of middle ear effusion.

PURPOSE: To further enhance and assess the ability to characterize middle ear effusion (MEE) using non-invasive ultrasound technology. MATERIALS AND METHODS: This is a prospective unblinded comparison study. Fifty-six children between the ages of 6 months and 17 years scheduled to undergo bilateral myringotomy with pressure equalization tube placement were enrolled. With the child anesthetized, the probe was placed into the external ear canal after sterile water was inserted. Ultrasound recordings of middle ear contents were analyzed by computer algorithm. Middle ear fluid was collected during myringotomy and analyzed for bacterial culture and viscosity. RESULTS: Ultrasound waveforms yielded a computer algorithm interpretation of middle ear contents in 66% of ears tested. When a result was obtained, the sensitivity and specificity for successfully characterizing middle ear fluid content as either void of fluid, thick fluid (mucoid), or thin fluid (serous or purulent) were at least 94%. Mucoid effusions had higher measured viscosity values (P=.002). Viscosity measures were compared to culture result, and those with low viscosity (thin consistency) had a higher likelihood of having a positive culture (P=.048). CONCLUSION: The device sensitivity and specificity for fluid detection were 94% or greater among interpretable waveforms (66% of those tested). Although this technology provides important information of the middle ear effusion presence and characteristic, further technological improvements are needed.

From mouse to human: Accessing the biochemistry of vision in vivo by two-photon excitation.

The eye is an ideal organ for imaging by a multi-photon excitation approach, because ocular tissues such as the sclera, cornea, lens and neurosensory retina, are highly transparent to infrared (IR) light. The interface between the retina and the retinal pigment epithelium (RPE) is especially informative, because it reflects the health of the visual (retinoid) cycle and its changes in response to external stress, genetic manipulations, and drug treatments. Vitamin A-derived retinoids, like retinyl esters, are natural fluorophores that respond to multi-photon excitation with near IR light, bypassing the filter-like properties of the cornea, lens, and macular pigments. Also, during natural aging some retinoids form bisretinoids, like diretinoid-pyridiniumethanolamine (A2E), that are highly fluorescent. These bisretinoids appear to be elevated concurrently with aging. Vitamin A-derived retinoids and bisretinoidss are detected by two-photon ophthalmoscopy (2PO), using a new class of light sources with adjustable spatial, temporal, and spectral properties. Furthermore, the two-photon (2P) absorption of IR light by the visual pigments in rod and cone photoreceptors can initiate visual transduction by cis-trans isomerization of retinal, enabling parallel functional studies. Recently we overcame concerns about safety, data interpretation and complexity of the 2P-based instrumentation, the major roadblocks toward advancing this modality to the clinic. These imaging and retina-function assessment advancements have enabled us to conduct the first 2P studies with humans.

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.

Rapid RGR-dependent visual pigment recycling is mediated by the RPE and specialized Müller glia.

In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.

Imaging of protein crystals with two-photon microscopy.

Second-order nonlinear optical imaging of chiral crystals (SONICC), which portrays second-harmonic generation (SHG) by noncentrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal-to-noise ratio. Here we report the incorporation of both SONICC and two-photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as ~10 µm in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG-silent protein crystals. Our experimental data suggest that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPEF. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with nondamaging infrared light in a single system makes the combination of SHG and intrinsic visible TPEF a powerful tool for nondestructive protein crystal identification and characterization during crystallization trials.

Retinyl ester storage particles (retinosomes) from the retinal pigmented epithelium resemble lipid droplets in other tissues.

Levels of many hydrophobic cellular substances are tightly regulated because of their potential cytotoxicity. These compounds tend to self-aggregate in cytoplasmic storage depots termed lipid droplets/bodies that have well defined structures that contain additional components, including cholesterol and various proteins. Hydrophobic substances in these structures become mobilized in a specific and regulated manner as dictated by cellular requirements. Retinal pigmented epithelial cells in the eye produce retinyl ester-containing lipid droplets named retinosomes. These esters are mobilized to replenish the visual chromophore, 11-cis-retinal, and their storage ensures proper visual function despite fluctuations in dietary vitamin A intake. But it remains unclear whether retinosomes are structures specific to the eye or similar to lipid droplets in other organs/tissues that contain substances other than retinyl esters. Thus, we initially investigated the production of these lipid droplets in experimental cell lines expressing lecithin:retinol acyltransferase, a key enzyme involved in formation of retinyl ester-containing retinosomes from all-trans-retinol. We found that retinosomes and oleate-derived lipid droplets form and co-localize concomitantly, indicating their intrinsic structural similarities. Next, we isolated native retinosomes from bovine retinal pigmented epithelium and found that their protein and hydrophobic small molecular constituents were similar to those of lipid droplets reported for other experimental cell lines and tissues. These unexpected findings suggest a common mechanism for lipid droplet formation that exhibits broad chemical specificity for the hydrophobic substances being stored.

Retinal cone and rod photoreceptor cells exhibit differential susceptibility to light-induced damage.

All-trans-retinal and its condensation-products can cause retinal degeneration in a light-dependent manner and contribute to the pathogenesis of human macular diseases such as Stargardt's disease and age-related macular degeneration. Although these toxic retinoid by-products originate from rod and cone photoreceptor cells, the contribution of each cell type to light-induced retinal degeneration is unknown. In this study, the primary objective was to learn whether rods or cones are more susceptible to light-induced, all-trans-retinal-mediated damage. Previously, we reported that mice lacking enzymes that clear all-trans-retinal from the retina, ATP-binding cassette transporter 4 and retinol dehydrogenase 8, manifested light-induced retinal dystrophy. We first examined early-stage age-related macular degeneration patients and found retinal degenerative changes in rod-rich rather than cone-rich regions of the macula. We then evaluated transgenic mice with rod-only and cone-like-only retinas in addition to progenies of such mice inbred with Rdh8(-/-) Abca4(-/-) mice. Of all these strains, Rdh8(-/-) Abca4(-/-) mice with a mixed rod-cone population showed the most severe retinal degeneration under regular cyclic light conditions. Intense light exposure induced acute retinal damage in Rdh8(-/-) Abca4(-/-) and rod-only mice but not cone-like-only mice. These findings suggest that progression of retinal degeneration in Rdh8(-/-) Abca4(-/-) mice is affected by differential vulnerability of rods and cones to light.

Seeing invisible light: 2-photon microperimetry to measure visual function.

Purpose: The accuracy of conventional visual function tests, which emit visible light, decreases in patients with corneal scars, cataracts, and vitreous hemorrhages. In contrast, infrared (IR) light exhibits greater tissue penetrance than visible light and is less susceptible to optical opacities. We therefore compared conventional visual function tests against infrared 2-phton microperimetry (2PM-IR) in a subject with a brunescent nuclear sclerotic and posterior subcapsular cataract before and after cataract surgery. Methods: Testing using infrared light microperimetry from a novel device (2PM-IR), visible light microperimetry from a novel device (2PM-Vis), conventional microperimetry, and the cone contrast threshold (CCT) test were performed before and after cataract surgery. Results: Retinal sensitivity assessed using 2PM-IR, 2PM-Vis, and cMP improved by 3.4 dB, 17.4 dB, and 18 dB, respectively. Cone contrast threshold testing improved for the S-cone, M-cone, and l-cone by 111, 14, and 30. Conclusions and Importance: 2PM-IR, unlike conventional visual function tests, showed minimal variability in retinal sensitivity before and after surgery. Thus, IR visual stimulation may provide a more accurate means of measuring neurosensory retinal function by circumventing optical media opacities, aiding in the diagnosis of early macular disease.