Individual Cones Research Articles

Automated identification of cone photoreceptors in adaptive optics optical coherence tomography images using transfer learning

Automated measurements of the human cone mosaic requires the identification of individual cone photoreceptors. The current gold standard, manual labeling, is a tedious process and can not be done in a clinically useful timeframe. As such, we present an automated algorithm for identifying cone photoreceptors in adaptive optics optical coherence tomography (AO-OCT) images. Our approach fine-tunes a pre-trained convolutional neural network originally trained on AO scanning laser ophthalmoscope (AO-SLO) images, to work on previously unseen data from a different imaging modality. On average, the automated method correctly identified 94% of manually labeled cones when compared to manual raters, from twenty different AO-OCT images acquired from five normal subjects. Voronoi analysis confirmed the general hexagonal-packing structure of the cone mosaic as well as the general cone density variability across portions of the retina. The consistency of our measurements demonstrates the high reliability and practical utility of having an automated solution to this problem.

Visible to near-infrared thermal radiation from nanostructured tungsten anntennas

We report thermal radiation antennas that sustain large emissivity at a broad range of wavelengths and angles, as well as maintaining their emissivity at high temperatures (up to 1300 K) under atmospheric pressure. Two-dimensional arrays composed of tungsten (W) cones with high aspect ratio were fabricated by laser interference lithography followed by deep reactive-ion etching, then optically characterized by measuring the broadband (λ = 500–2500 nm) absorptivity spectra. The fabricated W radiation antennas yielded greater absorptivity in visible to near-infrared wavelengths by increasing the aspect ratio of the cones, resulting in a near two-fold enhancement in average absorptivity compared to a reference planar W substrate. The measured absorptivity spectra were reproduced well by electromagnetic simulations with the experimental optical constants of W. Electromagnetic simulations also verified that such broadband increases in absorptivity are mostly caused by the enlarged scattering cross-section of individual cones. A 120 nm-thick conformal alumina coating prepared by atomic layer deposition was employed to prevent surface oxidation of the fabricated W radiation antennas, which additionally improved their absorptivity through an amplified antenna effect. These experimental and theoretical findings are generalizable to different infrared spectra by simply scaling up the antenna structures, and thus will be extensively utilized in thermal radiation applications such as solar steamers, thermophotovoltaics, and radiative coolers.

Sensations from a single M-cone depend on the activity of surrounding S-cones

Color vision requires the activity of cone photoreceptors to be compared in post-receptoral circuitry. Decades of psychophysical measurements have quantified the nature of these comparative interactions on a coarse scale. How such findings generalize to a cellular scale remains unclear. To answer that question, we quantified the influence of surrounding light on the appearance of spots targeted to individual cones. The eye’s aberrations were corrected with adaptive optics and retinal position was precisely tracked in real-time to compensate for natural movement. Subjects reported the color appearance of each spot. A majority of L-and M-cones consistently gave rise to the sensation of white, while a smaller group repeatedly elicited hue sensations. When blue sensations were reported they were more likely mediated by M- than L-cones. Blue sensations were elicited from M-cones against a short-wavelength light that preferentially elevated the quantal catch in surrounding S-cones, while stimulation of the same cones against a white background elicited green sensations. In one of two subjects, proximity to S-cones increased the probability of blue reports when M-cones were probed. We propose that M-cone increments excited both green and blue opponent pathways, but the relative activity of neighboring cones favored one pathway over the other.

Cone synapses in mammalian retinal rod bipolar cells.

Some mammalian rod bipolar cells (RBCs) can receive excitatory chemical synaptic inputs from both rods and cones (DBCR2 ), but anatomical evidence for mammalian cone-RBC contacts has been sparse. We examined anatomical cone-RBC contacts using neurobiotin (NB) to visualize individual mouse cones and standard immuno-markers to identify RBCs, cone pedicles and synapses in mouse and baboon retinas. Peanut agglutinin (PNA) stained the basal membrane of all cone pedicles, and mouse cones were positive for red/green (R/G)-opsin, whereas baboon cones were positive for calbindin D-28k. All synapses in the outer plexiform layer were labeled for synaptic vesicle protein 2 (SV2) and PSD (postsynaptic density)-95, and those that coincided with PNA resided closest to bipolar cell somas. Cone-RBC synaptic contacts were identified by: (a) RBC dendrites deeply invaginating into the center of cone pedicles (invaginating synapses), (b) RBC dendritic spines intruding into the surface of cone pedicles (superficial synapses), and (c) PKCα immunoreactivity coinciding with synaptic marker SV2, PSD-95, mGluR6, G protein beta 5 or PNA at cone pedicles. One RBC could form 0-1 invaginating and 1-3 superficial contacts with cones. 20.7% and 38.9% of mouse RBCs contacted cones in the peripheral and central retina (p < .05, n = 14 samples), respectively, while 34.4% (peripheral) and 48.5% (central) of cones contacted RBCs (p > .05). In baboon retinas (n = 4 samples), cone-RBC contacts involved 12.2% of RBCs (n = 416 cells) and 22.5% of cones (n = 225 cells). This suggests that rod and cone signals in the ON pathway are integrated in some RBCs before reaching AII amacrine cells.

Multibands tunneling in AAA-stacked trilayer graphene

We study the electronic transport through np and npn junctions for AAA-stacked trilayer graphene. Two kinds of gates are considered where the first is a single gate and the second is a double gate. After obtaining the solutions for the energy spectrum, we use the transfer matrix method to determine the three transmission probabilities for each individual cone τ=0,±1. We show that the quasiparticles in AAA-stacked trilayer graphene are not only chiral but also labeled by an additional cone index τ. The obtained bands are composed of three Dirac cones that depend on the chirality indexes. We show that there is perfect transmission for normal or near normal incidence, which is a manifestation of the Klein tunneling effect. We analyze also the corresponding total conductance, which is defined as the sum of the conductance channels in each individual cone. Our results are numerically discussed and compared with those obtained for ABA- and ABC-stacked trilayer graphene.

A microfabricated on-chip approach to the micropipette growth cone-turning assay

Chemical gradients and physical contact with the surrounding microenvironment play an important role in regulating axonal pathfinding. Traditional experiments for chemically-induced growth cone-turning in vitro involve micropipettes filled with a guidance-cue solution. The localized chemical gradient generated through gravity-induced or pulsatile pressure application is used to evaluate the response of a single growth cone to the guidance-cue solution. Thus, the micropipette growth cone-turning assay has intrinsic application deficits that limit the duration of signaling. Moreover, it does not account for spatial reproducibility. To improve spatial resolution and expose multiple individual growth cones to identical environmental conditions, we developed a biochip system that incorporates geometric and chemical signaling to regulate axonal pathfinding. With this system, we were able to passively generate chemical gradients for long periods of time (>1 d) at continual flowrates of ∼40 pL s−1. Over the course of 4 DIV, angular distributions for axonal pathfinding of individual laser cell-patterned CFNs showed statistically different frequencies when a chemical guidance gradient was generated from a localized source. The biochip described here can be used to systematically test various chemical guidance molecules with high repeatability and longer durations.

Local Signals in Mouse Horizontal Cell Dendrites

The mouse retina contains a single type of horizontal cell, a GABAergic interneuron that samples from all cone photoreceptors within reach and modulates their glutamatergic output via parallel feedback mechanisms. Because horizontal cells form an electrically coupled network, they have been implicated in global signal processing, such as large-scale contrast enhancement. Recently, it has been proposed that horizontal cells can also act locally at the level of individual cone photoreceptors. To test this possibility physiologically, we used two-photon microscopy to record light stimulus-evoked Ca2+ signals in cone axon terminals and horizontal cell dendrites as well as glutamate release in the outer plexiform layer. By selectively stimulating the two mouse cone opsins with green and UV light, we assessed whether signals from individual cones remain isolated within horizontal cell dendritic tips or whether they spread across the dendritic arbor. Consistent with the mouse's opsin expression gradient, we found that the Ca2+ signals recorded from dendrites of dorsal horizontal cells were dominated by M-opsin and those of ventral horizontal cells by S-opsin activation. The signals measured in neighboring horizontal cell dendritic tips varied markedly in their chromatic preference, arguing against global processing. Rather, our experimental data and results from biophysically realistic modeling support the idea that horizontal cells can process cone input locally, extending the classical view of horizontal cell function. Pharmacologically removing horizontal cells from the circuitry reduced the sensitivity of the cone signal to low frequencies, suggesting that local horizontal cell feedback shapes the temporal properties of cone output.

Non-invasive assessment of human cone photoreceptor function.

Vision begins when light isomerizes the photopigments within photoreceptors. Noninvasive cellular-scale observation of the structure of the human photoreceptor mosaic is made possible through the use of adaptive optics (AO) enhanced ophthalmoscopes, but establishing noninvasive objective measures of photoreceptor function on a similar scale has been more difficult. AO ophthalmoscope images acquired with near-infrared light show that individual cone photoreceptor reflectance can change in response to a visible stimulus. Here we show that the intrinsic response depends on stimulus wavelength and intensity, and that its action spectrum is well-matched to the spectral sensitivity of cone-mediated vision. Our results demonstrate that the cone reflectance response is mediated by photoisomerization, thus making it a direct measure of photoreceptor function.

A method to quantify the "cone of economy".

A non-randomized, prospective, concurrent control cohort study. The purpose of this study is to develop and evaluate a method to quantify the dimensions of the cone of economy (COE) and the energy expenditure associated with maintaining a balanced posture within the COE, scoliosis patients and compare them to matched non-scoliotic controls in a group of adult degenerative. Balance is defined as the ability of the human body to maintain its center of mass (COM) within the base of support with minimal postural sway. The cone of economy refers to the stable region of upright standing posture. The underlying assumption is that deviating outside one's individual cone challenges the balance mechanisms. Adult degenerative scoliosis (ADS) patients exhibit a variety of postural changes within their COE, involving the spine, pelvis and lower extremities, in their effort to compensate for the altered posture. Ten ADS patients and ten non-scoliotic volunteers performed a series of functional balance tests. The dimensions of the COE and the energy expenditure related to maintaining balance within the COE were measured using a human motion video capture system and dynamic surface electromyography. ADS patients presented more COM sway in the sagittal (ADS: 1.59 cm vs. H: 0.61 cm; p = 0.049) and coronal (ADS: 2.84 cm vs. H: 1.72 cm; p = 0.046) directions in comparison to the non-scoliotic control. ADS patients presented with more COM (ADS: 33.30 cm vs. H: 19.13 cm; p = 0.039) and head (ADS: 31.06 cm vs. H: 19.13 cm; p = 0.013) displacements in comparison to the non-scoliotic controls. Scoliosis patients expended more muscle activity to maintain static standing, as manifest by increased muscle activity in their erector spinae (ADS: 37.16mV vs. H: 20.31mV; p=0.050), and gluteus maximus (ADS: 33.12mV vs. H: 12.09mV; p=0.001) muscles. We were able to develop and evaluate a method that quantifies the COE boundaries, COM displacement, and amount of sway within the COE along with the energy expenditure for a specific patient. This method of COE measurement will enable spine care practitioners to objectively evaluate their patients in an effort to determine the most appropriate treatment options, and in objectively documenting the effectiveness of their intervention.

Spatiochromatic Interactions between Individual Cone Photoreceptors in the Human Retina.

A remarkable feature of human vision is that the retina and brain have evolved circuitry to extract useful spatial and spectral information from signals originating in a photoreceptor mosaic with trichromatic constituents that vary widely in their relative numbers and local spatial configurations. A critical early transformation applied to cone signals is horizontal-cell-mediated lateral inhibition, which imparts a spatially antagonistic surround to individual cone receptive fields, a signature inherited by downstream neurons and implicated in color signaling. In the peripheral retina, the functional connectivity of cone inputs to the circuitry that mediates lateral inhibition is not cone-type specific, but whether these wiring schemes are maintained closer to the fovea remains unsettled, in part because central retinal anatomy is not easily amenable to direct physiological assessment. Here, we demonstrate how the precise topography of the long (L)-, middle (M)-, and short (S)-wavelength-sensitive cones in the human parafovea (1.5° eccentricity) shapes perceptual sensitivity. We used adaptive optics microstimulation to measure psychophysical detection thresholds from individual cones with spectral types that had been classified independently by absorptance imaging. Measured against chromatic adapting backgrounds, the sensitivities of L and M cones were, on average, receptor-type specific, but individual cone thresholds varied systematically with the number of preferentially activated cones in the immediate neighborhood. The spatial and spectral patterns of these interactions suggest that interneurons mediating lateral inhibition in the central retina, likely horizontal cells, establish functional connections with L and M cones indiscriminately, implying that the cone-selective circuitry supporting red-green color vision emerges after the first retinal synapse.SIGNIFICANCE STATEMENT We present evidence for spatially antagonistic interactions between individual, spectrally typed cones in the central retina of human observers using adaptive optics. Using chromatic adapting fields to modulate the relative steady-state activity of long (L)- and middle (M)-wavelength-sensitive cones, we found that single-cone detection thresholds varied predictably with the spectral demographics of the surrounding cones. The spatial scale and spectral pattern of these photoreceptor interactions were consistent with lateral inhibition mediated by retinal horizontal cells that receive nonselective input from L and M cones. These results demonstrate a clear link between the neural architecture of the visual system inputs-cone photoreceptors-and visual perception and have implications for the neural locus of the cone-specific circuitry supporting color vision.

The spatial structure of cone-opponent receptive fields in macaque retina

The receptive field structure of long (L) to middle (M) wavelength (L/M) cone-opponent ganglion cells of the parafoveal macaque retina was investigated using drifting gratings. Gratings were luminance, chromatic or selective for the L- or M-cones. Based on these spatial tuning curves, receptive field profiles for the individual cones were derived. Receptive field profiles were coarse compared to single cones, and often could not be described by a simple Gaussian, having shallower flanks. There was a continuum of spatial properties, which blurred any systematic distinction between Type I and Type II receptive fields. Opponent center-surround organization within a single cone was rare. Usually, responses to all four grating types could be described based on the cone receptive field profiles. An exception was a few cells that showed irregularities of amplitude and phase at high spatial frequencies for one or other of the cone isolating conditions. The data are related to standard models of M/L opponent receptive fields and implications for central processing are considered.

Synaptic Ribbon Active Zones in Cone Photoreceptors Operate Independently from One Another.

Cone photoreceptors depolarize in darkness to release glutamate-laden synaptic vesicles. Essential to release is the synaptic ribbon, a structure that helps organize active zones by clustering vesicles near proteins that mediate exocytosis, including voltage-gated Ca2+ channels. Cone terminals have many ribbon-style active zones at which second-order neurons receive input. We asked whether there are functionally significant differences in local Ca2+ influx among ribbons in individual cones. We combined confocal Ca2+ imaging to measure Ca2+ influx at individual ribbons and patch clamp recordings to record whole-cell ICa in salamander cones. We found that the voltage for half-maximal activation (V50) of whole cell ICa in cones averaged −38.1 mV ± 3.05 mV (standard deviation [SD]), close to the cone membrane potential in darkness of ca. −40 mV. Ca2+ signals at individual ribbons varied in amplitude from one another and showed greater variability in V50 values than whole-cell ICa, suggesting that Ca2+ signals can differ significantly among ribbons within cones. After accounting for potential sources of technical variability in measurements of Ca2+ signals and for contributions from cone-to-cone differences in ICa, we found that the variability in V50 values for ribbon Ca2+ signals within individual cones showed a SD of 2.5 mV. Simulating local differences in Ca2+ channel activity at two ribbons by shifting the V50 value of ICa by ±2.5 mV (1 SD) about the mean suggests that when the membrane depolarizes to −40 mV, two ribbons could experience differences in Ca2+ influx of >45%. Further evidence that local Ca2+ changes at ribbons can be regulated independently was obtained in experiments showing that activation of inhibitory feedback from horizontal cells (HCs) to cones in paired recordings changed both amplitude and V50 of Ca2+ signals at individual ribbons. By varying the strength of synaptic output, differences in voltage dependence and amplitude of Ca2+ signals at individual ribbons shape the information transmitted from cones to downstream neurons in vision.

Principles of Adaptive Optics and Clinical Applications

The human retina is a uniquely accessible tissue. Tools like scanning laser ophthalmoscopy and spectral domain optical coherence tomography provide clinicians with remarkably clear pictures of the living retina. While the anterior optics of the eye permit such non-invasive visualization of the retina and associated pathology, these same optics induce significant aberrations that in most cases obviate cellular-resolution imaging. Adaptive optics (AO) imaging systems use active optical elements to compensate for aberrations in the optical path between the object and the camera. Applied to the human eye, AO allows direct visualization of individual rod and cone photoreceptor cells, Retina pigment epithelial cells and white blood cells. AO imaging has changed the way vision scientists and ophthalmologists see the retina, helping to clarify our understanding of retinal structure, function and the etiology of various retinal pathologies. Here we review some of the advances made possible with AO imaging of the human retina and discuss applications and future prospects for clinical imaging.

Local signal processing in mouse horizontal cell dendrites

The mouse retina contains a single type of horizontal cell, a GABAergic interneuron that samples from all cone photoreceptors within reach and modulates their glutamatergic output via parallel feedback mechanisms. Because horizontal cells form an electrically-coupled network, they have been implicated in global signal processing, such as large scale contrast enhancement. Recently, it has been proposed that horizontal cells can also act locally at the level of individual cone photoreceptor axon terminals. To test this possibility physiologically, we used two-photon microscopy to record light-evoked Ca2+ signals in cone axon terminals and horizontal cell dendrites as well as glutamate release in the outer plexiform layer. By selectively stimulating the two mouse cone opsins with green and UV light, we assessed whether signals from individual cones remain “isolated” within horizontal cell dendritic tips, or whether they spread across the dendritic arbour. Consistent with the mouse9s opsin expression gradient, we found that light responses recorded from dendrites of dorsal horizontal cells were dominated by M- and those of ventral horizontal cells by S-opsin activation. Light responses measured in neighbouring horizontal cell dendritic tips varied markedly in their chromatic preference, arguing against global processing. Rather, our data support the idea that horizontal cells can process cone input locally, extending the “classical” view of horizontal cells function. Pharmacologically removing horizontal cells from the circuitry reduced the sensitivity of the cone signal to low frequencies, suggesting that local horizontal cell feedback shapes the temporal properties of cone output.

Determination of the Genetic Architecture Underlying Short Wavelength Sensitivity in Lake Malawi Cichlids.

African cichlids are an exemplary system to study organismal diversity and rapid speciation. Species differ in external morphology including jaw shape and body coloration, but also differ in sensory systems including vision. All cichlids have 7 cone opsin genes with species differing broadly in which opsins are expressed. The differential opsin expression results in closely related species with substantial differences in spectral sensitivity of their photoreceptors. In this work, we take a first step in determining the genetic basis of opsin expression in cichlids. Using a second generation cross between 2 species with different opsin expression patterns, we make a conservative estimate that short wavelength opsin expression is regulated by a few loci. Genetic mapping in 96 F2 hybrids provides clear evidence of a cis-regulatory region for SWS1 opsin that explains 34% of the variation in expression between the 2 species. Additionally, in situ hybridization has shown that SWS1 and SWS2B opsins are coexpressed in individual single cones in the retinas of F2 progeny. Results from this work will contribute to a better understanding of the genetic architecture underlying opsin expression. This knowledge will help answer long-standing questions about the evolutionary processes fundamental to opsin expression variation and how this contributes to adaptive cichlid divergence.

Growth of Stone pine (Pinus pinea L.) European provenances in central Chile

Abstract: Pinus pinea is characterized by phenotypic plasticity, tolerance to harsh soils and climates, but low differentiation in growth parameters and low genetic variability. Growth and cone production of six European stone pine provenances (two from Italy, three from Spain and one from Slovenia) were analyzed in a field trial experiment established in central Chile. The study evaluated height, diameter at breast height (DBH) and crown diameter growth of 147 nineteen-year-old trees per provenance, as well as fruiting variables (i.e., number of cones per tree and cone weight). Survival over the first 7 years was also evaluated. Provenances significantly differed in cone number per tree, cone weight, height and DBH growth, and crown diameter growth. Provenances were grouped according to growth and production variables: one group included the Italian and Slovenian provenances, the second group Andalucia and Sierra Morena (Spain), and the third included Meseta Castellana (Spain). Individual cone production was positively correlated with cone weight and other growth variables. Meseta Castellana provenance showed the highest growth and productivity. Our results provide useful information for the selection of P. pinea provenances to be used in new plantations in central Chile.

Low turn-on field nanodiamond conic field emitter

Field emission properties of individual nanocrystalline diamond cones with higher sp2 content were investigated using a double-probe scanning electron microscope system. The nanodiamond cones were fabricated utilizing gray-scale patterns with a focused-ion-beam (FIB) system. The as-formed nanocones with slicing number of 150 have high content of sp2 amorphous carbon surfaces and thus show a high emission current up to 54μA at an applied voltage of 10V. In addition, twice higher emission current was achieved by UV illumination under even lower turn-on field (1.1V/μm), which benefits the realization of high performance cold cathode field emitters and UV detectors using such nanostructures.

Validation of a Cochlear Implant Patient-Specific Model of the Voltage Distribution in a Clinical Setting.

Cochlear Implants (CIs) are medical implantable devices that can restore the sense of hearing in people with profound hearing loss. Clinical trials assessing speech intelligibility in CI users have found large intersubject variability. One possibility to explain the variability is the individual differences in the interface created between electrodes of the CI and the auditory nerve. In order to understand the variability, models of the voltage distribution of the electrically stimulated cochlea may be useful. With this purpose in mind, we developed a parametric model that can be adapted to each CI user based on landmarks from individual cone beam computed tomography (CBCT) scans of the cochlea before and after implantation. The conductivity values of each cochlea compartment as well as the weighting factors of different grounding modes have also been parameterized. Simulations were performed modeling the cochlea and electrode positions of 12 CI users. Three models were compared with different levels of detail: a homogeneous model (HM), a non-patient-specific model (NPSM), and a patient-specific model (PSM). The model simulations were compared with voltage distribution measurements obtained from the backward telemetry of the 12 CI users. Results show that the PSM produces the lowest error when predicting individual voltage distributions. Given a patient-specific geometry and electrode positions, we show an example on how to optimize the parameters of the model and how to couple it to an auditory nerve model. The model here presented may help to understand speech performance variability and support the development of new sound coding strategies for CIs.

Photoreceptor disc shedding in the living human eye.

Cone photoreceptors undergo a daily cycle of renewal and shedding of membranous discs in their outer segments (OS), the portion responsible for light capture. These physiological processes are fundamental to maintaining photoreceptor health, and their dysfunction is associated with numerous retinal diseases. While both processes have been extensively studied in animal models and postmortem eyes, little is known about them in the living eye, in particular human. In this study, we report discovery of the optical signature associated with disc shedding using a method based on adaptive optics optical coherence tomography (AO-OCT) in conjunction with post-processing methods to track and monitor individual cone cells in 4D. The optical signature of disc shedding is characterized by an abrupt transient loss in the cone outer segment tip (COST) reflection followed by its return that is axially displaced anteriorly. Using this signature, we measured the temporal and spatial properties of shedding events in three normal subjects. Average duration of the shedding event was 8.8 ± 13.4 minutes, and average length loss of the OS was 2.1 μm (7.0% of OS length). Prevalence of cone shedding was highest in the morning (14.3%) followed by the afternoon (5.7%) and evening (4.0%), with load distributed across the imaged patch. To the best of our knowledge these are the first images of photoreceptor disc shedding in the living retina.

Viability and germination of Scots pine seeds after freezing of harvested cones in vitro

Scots pine (Pinus silvestris L.) cone and seed water contents were analyzed in two consecutive seasons during maturation stage in the autumn and in January and March before seed dispersal. Cones with different water contents were subjected to 2 h of freezing at −30 °C, and seed viability and laboratory germination of seeds from individual cones after treatment were analyzed. Seed water content could be well predicted with the measurement of the cone water content, and the general relationship between these two could be described with a generalized logistic function. On average, the water content of cones was 5%–10% units higher than the seeds inside them. The higher the cone water content at the onset of freezing treatment, the higher the proportion of seeds with apparent damage (based on visual inspection of seeds using X-ray images) in that particular cone. High water content in cones also resulted in decreased germination after freezing treatment. The critical cone water content for 50% germination after freezing at −30 °C was approximately 31.3% (fresh mass basis). This corresponds to 21.6% water content in seeds.