Visual and Physiological Optics: introduction to the joint feature issue in Biomedical Optics Express and Journal of the Optical Society of America A.

This feature issue collects articles presented at the tenth Visual and Physiological Optics meeting (VPO2022), held August 29-31, 2022, in Cambridge, UK. This joint feature issue between Biomedical Optics Express and Journal of the Optical Society of America A includes articles that cover the broad range of topics addressed at the meeting and examples of the current state of research in the field.

Introduction to Visual and Physiological Optics feature issue of Biomedical Optics Express and JOSA A.

This feature issue collects articles presented at the tenth Visual and Physiological Optics meeting (VPO2022), held August 29-31, 2022, in Cambridge, UK. This joint feature issue between Biomedical Optics Express and Journal of the Optical Society of America A includes articles that cover the broad range of topics addressed at the meeting and examples of the current state of research in the field.

SLM-based interferometer for assessing the polychromatic neural transfer function of the eye.

A novel interferometric instrument for measuring neural transfer function (NTF) of the eye is presented. The device is based on a liquid-crystal-on-silicon spatial light modulator (SLM), which is used to create two laterally separated wavefronts in the pupil plane of the eye that interfere on the retina. The phase mask on the SLM, consisting of two diffraction gratings mixed in a checkerboard pattern and acting as a shearing interferometer, allows independent control of spatial frequency, orientation, and contrast of the fringes, as well as the field of view in a wide polychromatic spectrum. Coupled with a supercontinuum source, the system is able to produce achromatic fringes on the retina. The instrument was successfully tested in six normal subjects in four light conditions: polychromatic light and monochromatic blue, green and red light respectively (central wavelengths - 450, 550 and 650 nm). On average, the NTF in polychromatic light was approximately 20% higher than for green and red light, although not statistically significant due to high intersubject variability. Due to all-digital control of the interference fringes, the device is optically simple and virtually unsusceptible to vibrations, allowing its use in a non-laboratory environment. The study also contributes to color vision research, allowing to evaluate contrast sensitivity function without monochromatic or chromatic aberrations.

Localized Photoreceptor Ablation Using Femtosecond Pulses Focused With Adaptive Optics.

Purpose: The development of new approaches to human vision restoration could be greatly accelerated with the use of nonhuman primate models; however, there is a paucity of primate models of outer retina degeneration with good spatial localization. To limit ablation to the photoreceptors, we developed a new approach that uses a near-infrared ultrafast laser, focused using adaptive optics, to concentrate light in a small focal volume within the retina. Methods: In the eyes of eight anesthetized macaques, 187 locations were exposed to laser powers from 50 to 210 mW. Laser exposure locations were monitored for up to 18 months using fluorescein angiography (FA), optical coherence tomography (OCT), scanning laser ophthalmoscopy (SLO), adaptive optics scanning laser ophthalmoscope (AOSLO) reflectance imaging, two-photon excited fluorescence (TPEF) ophthalmoscopy, histology, and calcium responses of retinal ganglion cells. Results: This method produced localized photoreceptor loss with minimal axial spread of damage to other retinal layers, verified by in-vivo structural imaging and histologic examination, although in some cases evidence of altered autofluorescence was found in the adjacent retinal pigment epithelium (RPE). Functional assessment using blood flow imaging of the retinal plexus and calcium imaging of the response of ganglion cells above the photoreceptor loss shows that inner retinal circuitry was preserved. Conclusions: Although different from a genetic model of retinal degeneration, this model of localized photoreceptor loss may provide a useful testbed for vision restoration studies in nonhuman primates. Translational Relevance: With this model, a variety of vision restoration methods can be tested in the non-human primate.

Visual and physiological optics: introduction to the Journal of the Optical Society of America A feature issue.

The 9th European Meeting on Visual Physiological Optics (VPO2018) was held August 29-31, 2018, in Athens, Greece. This issue of the Journal of the Optical Society of America A (JOSA A) is a dedicated feature, including numerous articles that span a large range of visual optics related topics, ranging from geometrical optics to visual psychophysics and from optical metrology to ophthalmic diagnostic technologies. The next VPO meeting will be held August 2020 in Cambridge, UK.

Cellular-scale evaluation of induced photoreceptor degeneration in the living primate eye.

Progress is needed in developing animal models of photoreceptor degeneration and evaluating such models with longitudinal, noninvasive techniques. We employ confocal scanning laser ophthalmoscopy, optical coherence tomography (OCT) and high-resolution retinal imaging to noninvasively observe the retina of non-human primates with induced photoreceptor degeneration. Photoreceptors were imaged at the single-cell scale in three modalities of adaptive optics scanning light ophthalmoscopy: traditional confocal reflectance, indicative of waveguiding; a non-confocal offset aperture technique visualizing scattered light; and two-photon excited fluorescence, the time-varying signal of which, at 730 nm excitation, is representative of visual cycle function. Assessment of photoreceptor structure and function using these imaging modalities revealed a reduction in retinoid production in cone photoreceptor outer segments while inner segments appeared to remain present. Histology of one retina confirmed loss of outer segments and the presence of intact inner segments. This unique combination of imaging modalities can provide essential, clinically-relevant information on both the structural integrity and function of photoreceptors to not only validate models of photoreceptor degeneration but potentially evaluate the efficacy of future cell and gene-based therapies for vision restoration.

Selective S Cone Damage and Retinal Remodeling Following Intense Ultrashort Pulse Laser Exposures in the Near-Infrared.

Purpose: Infrared ultrashort pulse lasers are becoming increasingly popular for applications in the living eye. However, safety standards are not yet well established. Here we investigate retinal damage close to threshold for this pulse regime in the living macaque eye. Methods: Retinal radiant exposures between 214 and 856 J/cm2 were delivered to the photoreceptor layer with an ultrashort pulse laser (730 nm, 55 fs, 80 MHz) through a two-photon adaptive optics scanning light ophthalmoscope. Retinal exposures were followed up immediately after and over several weeks with high-resolution reflectance and two-photon excited fluorescence ophthalmoscopy, providing structural and functional information. Results: Retinal radiant exposures of 856 J/cm2 resulted in permanent S cone damage. Immediately after the exposure, the affected cones emitted about 2.6 times less two-photon excited fluorescence (TPEF) and showed an altered TPEF time course. Several weeks after the initial exposure, S cone outer and inner segments had disappeared. The space was filled by rods in the peripheral retina and cones near the fovea. Conclusion: Interestingly, S cones are the receptor class with the lowest sensitivity in the near-infrared but are known to be particularly susceptible to ultraviolet and blue light. This effect of selective S cone damage after intense infrared ultrashort pulse laser exposure may be due to nonlinear absorption and distinct from pure thermal and mechanical mechanisms often associated with ultrashort pulse lasers.

Engaging Undergraduate Biology Students in Scientific Modeling: Analysis of Group Interactions, Sense-Making, and Justification.

National calls for improving science education (e.g., Vision and Change) emphasize the need to learn disciplinary core ideas through scientific practices. To address this need, we engaged small groups of students in developing diagrammatic models within two (one large-enrollment and one medium-enrollment) undergraduate introductory biology courses. During these activities, students developed scientific models of biological phenomena such as enhanced growth in genetically modified fish. To investigate whether undergraduate students productively engaged in scientific practices during these modeling activities, we recorded groups of students as they developed models and examined three characteristics: how students 1) interacted with one another, 2) made sense of phenomena, and 3) justified their ideas. Our analysis indicates that students spent most of the time on task, developing and evaluating their models. Moreover, they worked cooperatively to make sense of core ideas and justified their ideas to one another throughout the activities. These results demonstrate that, when provided with the opportunity to develop models during class, students in large-enrollment lecture courses can productively engage in scientific practices. We discuss potential reasons for these outcomes and suggest areas of future research to continue advancing knowledge regarding engaging students in scientific practices in large-enrollment lecture courses.

Mechanical and biological effects of infiltration with biopolymers on 3D printed tricalciumphosphate scaffolds.

The aim of this study was to evaluate the influence of infiltrating 3D printed (TCP) scaffolds with different biodegradable polymers on their mechanical and biological properties. 3D printed TCP scaffolds with interconnecting channels measuring 450±50 µm were infiltrated with four different biodegradable copolymers. To determine the average compressive strength, a uniaxial testing system was used. Additionally, scaffolds were seeded with MC3T3 cells and cell viability was assessed by live/dead-assay. Uninfiltrated TCP had an average compression strength of 1.92±0.38 MPa. Mechanical stability was considerably increased in all infiltrated scaffolds up to a maximum of 7.36±0.57 MPa. All scaffolds demonstrated high cell survival rates with a maximum of 94±10 % living cells. In conclusion, infiltration of 3D printed tricalcium phosphate scaffolds with biodegradable polymers significantly improved mechanical properties and biological properties were comparable to those of uninfiltrated TCP scaffolds.

Imaging individual neurons in the retinal ganglion cell layer of the living eye.

Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Formation and Clearance of All-Trans-Retinol in Rods Investigated in the Living Primate Eye With Two-Photon Ophthalmoscopy.

Purpose: Two-photon excited fluorescence (TPEF) imaging has potential as a functional tool for tracking visual pigment regeneration in the living eye. Previous studies have shown that all-trans-retinol is likely the chief source of time-varying TPEF from photoreceptors. Endogenous TPEF from retinol could provide the specificity desired for tracking the visual cycle. However, in vivo characterization of native retinol kinetics is complicated by visual stimulation from the imaging beam. We have developed an imaging scheme for overcoming these challenges and monitored the formation and clearance of retinol. Methods: Three macaques were imaged by using an in vivo two-photon ophthalmoscope. Endogenous TPEF was excited at 730 nm and recorded through the eye's pupil for more than 90 seconds. Two-photon excited fluorescence increased with onset of light and plateaued within 40 seconds, at which point, brief incremental stimuli were delivered at 561 nm. The responses of rods to stimulation were analyzed by using first-order kinetics. Results: Two-photon excited fluorescence resulting from retinol production corresponded to the fraction of rhodopsin bleached. The photosensitivity of rhodopsin was estimated to be 6.88 ± 5.50 log scotopic troland. The rate of retinol clearance depended on intensity of incremental stimulation. Clearance was faster for stronger stimuli and time constants ranged from 50 to 300 seconds. Conclusions: This study demonstrates a method for rapidly measuring the rate of clearance of retinol in vivo. Moreover, TPEF generated due to retinol can be used as a measure of rhodopsin depletion, similar to densitometry. This enhances the utility of two-photon ophthalmoscopy as a technique for evaluating the visual cycle in the living eye.

Safety assessment in macaques of light exposures for functional two-photon ophthalmoscopy in humans.

Two-photon ophthalmoscopy has potential for in vivo assessment of function of normal and diseased retina. However, light safety of the sub-100 fs laser typically used is a major concern and safety standards are not well established. To test the feasibility of safe in vivo two-photon excitation fluorescence (TPEF) imaging of photoreceptors in humans, we examined the effects of ultrashort pulsed light and the required light levels with a variety of clinical and high resolution imaging methods in macaques. The only measure that revealed a significant effect due to exposure to pulsed light within existing safety standards was infrared autofluorescence (IRAF) intensity. No other structural or functional alterations were detected by other imaging techniques for any of the exposures. Photoreceptors and retinal pigment epithelium appeared normal in adaptive optics images. No effect of repeated exposures on TPEF time course was detected, suggesting that visual cycle function was maintained. If IRAF reduction is hazardous, it is the only hurdle to applying two-photon retinal imaging in humans. To date, no harmful effects of IRAF reduction have been detected.

Night myopia is reduced in binocular vision.

Night myopia, which is a shift in refraction with light level, has been widely studied but still lacks a complete understanding. We used a new infrared open-view binocular Hartmann-Shack wave front sensor to quantify night myopia under monocular and natural binocular viewing conditions. Both eyes' accommodative response, aberrations, pupil diameter, and convergence were simultaneously measured at light levels ranging from photopic to scotopic conditions to total darkness. For monocular vision, reducing the stimulus luminance resulted in a progression of the accommodative state that tends toward the subject's dark focus or tonic accommodation and a change in convergence following the induced accommodative error. Most subjects presented a myopic shift of accommodation that was mitigated in binocular vision. The impact of spherical aberration on the focus shift was relatively small. Our results in monocular conditions support the hypothesis that night myopia has an accommodative origin as the eye progressively changes its accommodation state with decreasing luminance toward its resting state in total darkness. On the other hand, binocularity restrains night myopia, possibly by using fusional convergence as an additional accommodative cue, thus reducing the potential impact of night myopia on vision at low light levels.

In Vivo Two-Photon Fluorescence Kinetics of Primate Rods and Cones.

PURPOSE: The retinoid cycle maintains vision by regenerating bleached visual pigment through metabolic events, the kinetics of which have been difficult to characterize in vivo. Two-photon fluorescence excitation has been used previously to track autofluorescence directly from retinoids and pyridines in the visual cycle in mouse and frog retinas, but the mechanisms of the retinoid cycle are not well understood in primates. METHODS: We developed a two-photon fluorescence adaptive optics scanning light ophthalmoscope dedicated to in vivo imaging in anesthetized macaques. Using pulsed light at 730 nm, two-photon fluorescence was captured from rods and cones during light and dark adaptation through the eye's pupil. RESULTS: The fluorescence from rods and cones increased with light exposure but at different rates. During dark adaptation, autofluorescence declined, with cone autofluorescence decreasing approximately 4 times faster than from rods. Rates of autofluorescence decrease in rods and cones were approximately 4 times faster than their respective rates of photopigment regeneration. Also, subsets of sparsely distributed cones were less fluorescent than their neighbors immediately following bleach at 565 nm and they were comparable with the S cone mosaic in density and distribution. CONCLUSIONS: Although other molecules could be contributing, we posit that these fluorescence changes are mediated by products of the retinoid cycle. In vivo two-photon ophthalmoscopy provides a way to monitor noninvasively stages of the retinoid cycle that were previously inaccessible in the living primate eye. This can be used to assess objectively photoreceptor function in normal and diseased retinas.

Binocular visual performance with aberration correction as a function of light level.

The extent to which monocular visual performance of subjects with normal amounts of ocular aberrations can be improved with adaptive optics (AO) depends on both the pupil diameter and the luminance for visual testing. Here, the benefit of correction of higher order aberrations for binocular visual performance was assessed over a range of luminances for natural light-adapted pupil sizes with a binocular AO visual simulator. Results show that binocular aberration correction benefits for visual acuity and contrast sensitivity increase with decreasing luminances. Also, the advantage of binocular over monocular viewing increases when visual acuity becomes worse. The findings suggest that binocular summation mitigates poor visual performance under low luminance conditions.

Comparison of binocular through-focus visual acuity with monovision and a small aperture inlay.

Corneal small aperture inlays provide extended depth of focus as a solution to presbyopia. As this procedure is becoming more popular, it is interesting to compare its performance with traditional approaches, such as monovision. Here, binocular visual acuity was measured as a function of object vergence in three subjects by using a binocular adaptive optics vision analyzer. Visual acuity was measured at two luminance levels (photopic and mesopic) under several optical conditions: 1) natural vision (4 mm pupils, best corrected distance vision), 2) pure-defocus monovision ( + 1.25 D add in the nondominant eye), 3) small aperture monovision (1.6 mm pupil in the nondominant eye), and 4) combined small aperture and defocus monovision (1.6 mm pupil and a + 0.75 D add in the nondominant eye). Visual simulations of a small aperture corneal inlay suggest that the device extends DOF as effectively as traditional monovision in photopic light, in both cases at the cost of binocular summation. However, individual factors, such as aperture centration or sensitivity to mesopic conditions should be considered to assure adequate visual outcomes.

Aged human mesenchymal stem cells: the duration of bone morphogenetic protein-2 stimulation determines induction or inhibition of osteogenic differentiation.

Bone morphogenetic protein 2 (BMP-2) is a potent osteoinductive cytokine and a growing number of in vitro studies analyze its effects on human mesenchymal stem cells (hMSC) derived from aged or osteoporotic donors. In these studies the exact quantification of osteogenic differentiation capacity is of fundamental interest. Nevertheless, the experimental conditions for osteogenic differentiation of aged hMSC have not been evaluated systematically and vary to a considerable extend. Aim of the study was to assess the influence of cell density, osteogenic differentiation media (ODM) change intervals and duration of BMP-2 stimulation on osteoinduction. Furthermore, time series were carried out for osteogenic differentiation and BMP-2 concentration in ODM/BMP-2 cell culture supernatants. The experiments were performed using hMSC isolated from femoral heads of aged patients undergoing hip joint replacement. ODM change intervals of 96 hours resulted in significantly higher calcium deposition compared to shorter intervals. A cell density of 80% prior to stimulation led to stronger osteoinduction compared to higher cell densities. In ODM, aged hMSC showed a significant induction of calcium deposition after 9 days. Added to ODM, BMP-2 showed a stable concentration in the cell culture supernatants for at least 96 hours. Addition of BMP-2 to ODM for the initial 4 days led to a significantly higher induction of osteogenic differentiation compared to ODM alone. On the other hand, addition of BMP-2 for 21 days almost abrogated the osteoinductive effect of ODM. We could demonstrate that the factors investigated have a substantial impact on the extent of osteogenic differentiation of aged hMSC. Consequently, it is of upmost importance to standardize the experimental conditions in order to enable comparability between different studies. We here define standard conditions for osteogenic differentiation in regard to the specific features of aged hMSC. The finding that BMP-2 induces or inhibits osteogenic differentiation in a time dependent manner indicates an age related alteration in signal transduction of hMSC and requires further investigation.

Binocular visual acuity for the correction of spherical aberration in polychromatic and monochromatic light.

Correction of spherical (SA) and longitudinal chromatic aberrations (LCA) significantly improves monocular visual acuity (VA). In this work, the visual effect of SA correction in polychromatic and monochromatic light on binocular visual performance is investigated. A liquid crystal based binocular adaptive optics visual analyzer capable of operating in polychromatic light is employed in this study. Binocular VA improves when SA is corrected and LCA effects are reduced separately and in combination, resulting in the highest value for SA correction in monochromatic light. However, the binocular summation ratio is highest for the baseline condition of uncorrected SA in polychromatic light. Although SA correction in monochromatic light has a greater impact monocularly than binocularly, bilateral correction of both SA and LCA may further improve binocular spatial visual acuity which may support the use of aspheric-achromatic ophthalmic devices, in particular, intraocular lenses (IOLs).

Mesenchymal stem cells from osteoporotic patients feature impaired signal transduction but sustained osteoinduction in response to BMP-2 stimulation.

Osteoporotic fractures show reduced callus formation and delayed bone healing. Cellular sources of fracture healing are mesenchymal stem cells (MSC) that differentiate into osteoblasts by stimulation with osteoinductive cytokines, such as BMP-2. We hypothesized that impaired signal transduction and reduced osteogenic differentiation capacity in response to BMP-2 may underlie the delayed fracture healing. Therefore, MSC were isolated from femoral heads of healthy and osteoporotic patients. Grouping was carried out by bone mineral densitometry in an age-matched manner. MSC were stimulated with BMP-2. Signal transduction was assessed by western blotting of pSMAD1/5/8 and pERK1/2 as well as by quantitative RT-PCR of Runx-2, Dlx5, and Osteocalcin. Osteogenic differentiation was assessed by quantifying Alizarin Red staining. Osteoporotic MSC featured an accurate phosphorylation pattern of SMAD1/5/8 but a significantly reduced activation of ERK1/2 by BMP-2 stimulation. Furthermore, osteoporotic MSC showed significantly reduced basal expression levels of Runx-2 and Dlx5. However, Runx-2, Dlx5, and Osteocalcin expression showed adequate up-regulation due to BMP-2 stimulation. The global osteogenic differentiation in standard osteogenic differentiation media was reduced in osteoporotic MSC. Nevertheless, osteoporotic MSC were shown to feature an adequate induction of osteogenic differentiation due to BMP-2 stimulation. Taken together, we here demonstrate osteoporosis associated alterations in BMP-2 signaling but sustained specific osteogenic differentiation capacity in response to BMP-2. Therefore, BMP-2 may represent a promising therapeutic agent for the treatment of fractures in osteoporotic patients.

Impact on stereo-acuity of two presbyopia correction approaches: monovision and small aperture inlay.

Some of the different currently applied approaches that correct presbyopia may reduce stereovision. In this work, stereo-acuity was measured for two methods: (1) monovision and (2) small aperture inlay in one eye. When performing the experiment, a prototype of a binocular adaptive optics vision analyzer was employed. The system allowed simultaneous measurement and manipulation of the optics in both eyes of a subject. The apparatus incorporated two programmable spatial light modulators: one phase-only device using liquid crystal on silicon technology for wavefront manipulation and one intensity modulator for controlling the exit pupils. The prototype was also equipped with a stimulus generator for creating retinal disparity based on two micro-displays. The three-needle test was programmed for characterizing stereo-acuity. Subjects underwent a two-alternative forced-choice test. The following cases were tested for the stimulus placed at distance: (a) natural vision; (b) 1.5 D monovision; (c) 0.75 D monovision; (d) natural vision and small pupil; (e) 0.75 D monovision and small pupil. In all cases the standard pupil diameter was 4 mm and the small pupil diameter was 1.6 mm. The use of a small aperture significantly reduced the negative impact of monovision on stereopsis. The results of the experiment suggest that combining micro-monovision with a small aperture, which is currently being implemented as a corneal inlay, can yield values of stereoacuity close to those attained under normal binocular vision.