Ametropia detection using a novel, compact wavefront autorefractor.

INTRODUCTION: Despite the well-known reproducibility issues of subjective refraction, most studies evaluating autorefractors compared differences between the device and subjective refraction. This work evaluated the performance of a novel handheld Hartmann-Shack-based autorefractor using an alternative protocol, which considered the inherent variability of subjective refraction. METHODS: Participants underwent an initial measurement with a desktop autorefractor, two subjective refractions (SR1 and SR2) and a final measurement with the QuickSee Free (QSFree) portable autorefractor. Autorefractor performance was evaluated by comparing the differences between the QSFree and each of the subjective refractions with the difference between the subjective refractions (SR1 vs. SR2) using Bland-Altman analysis and percentage of agreement. RESULTS: A total of 75 subjects (53 ± 14 years) were enrolled in the study. The average difference in the absolute spherical equivalent (M) between the QSFree and the SR1 and SR2 was ±0.24 and ±0.02 D, respectively, that is, very similar or smaller than the SR1 versus SR2 difference (±0.26 D). Average differences in astigmatic components were found to be negligible. The results demonstrate that differences between QSFree and both subjective refractions in J0 and J45 were within ±0.50 D for at least 96% of the measurements. The limits of agreement (LOAs) of the differences between QSFree and SR1, as well as QSFree and SR2, were higher than those observed between SR1 and SR2 for M, J0 and J45 . CONCLUSIONS: A protocol was designed and validated for the evaluation of a refractive device to account for the variability of subjective refraction. This protocol was used to evaluate a novel portable autorefractor and observed a smaller difference between the device and subjective refractions than the difference between the two subjective refraction measurements in terms of mean bias error, although the standard deviation was higher.

AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice.

BACKGROUND: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice. METHODS: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model. FINDINGS: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model. INTERPRETATION: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections. FUNDING: Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.

Multiplex viral tropism assay in complex cell populations with single-cell resolution.

Gene therapy constitutes one of the most promising mode of disease treatments. Two key properties for therapeutic delivery vectors are its transduction efficiency (how well the vector delivers therapeutic cargo to desired target cells) and specificity (how well it avoids off-target delivery into unintended cells within the body). Here we developed an integrated bioinformatics and experimental pipeline that enables multiplex measurement of transduction efficiency and specificity, particularly by measuring how libraries of delivery vectors transduce libraries of diverse cell types. We demonstrated that pairing high-throughput measurement of AAV identity with high-resolution single-cell RNA transcriptomic sequencing maps how natural and engineered AAV variants transduce individual cells within human cerebral and ocular organoids. We further demonstrate that efficient AAV transduction observed in organoids is recapitulated in vivo in non-human primates. This library-on-library technology will be important for determining the safety and efficacy of therapeutic delivery vectors.

Correction: Assesment of the QuickSee wavefront autorefractor for characterizing refractive errors in school-age children.

[This corrects the article DOI: 10.1371/journal.pone.0240933.].

Prediction of manifest refraction using machine learning ensemble models on wavefront aberrometry data.

PURPOSE: To assess the performance of machine learning (ML) ensemble models for predicting patient subjective refraction (SR) using demographic factors, wavefront aberrometry data, and measurement quality related metrics taken with a low-cost portable autorefractor. METHODS: Four ensemble models were evaluated for predicting individual power vectors (M, J0, and J45) corresponding to the eyeglass prescription of each patient. Those models were random forest regressor (RF), gradient boosting regressor (GB), extreme gradient boosting regressor (XGB), and a custom assembly model (ASB) that averages the first three models. Algorithms were trained on a dataset of 1244 samples and the predictive power was evaluated with 518 unseen samples. Variables used for the prediction were age, gender, Zernike coefficients up to 5th order, and pupil related metrics provided by the autorefractor. Agreement with SR was measured using Bland-Altman analysis, overall prediction error, and percentage of agreement between the ML predictions and subjective refractions for different thresholds (0.25 D, 0.5 D). RESULTS: All models considerably outperformed the predictions from the autorefractor, while ASB obtained the best results. The accuracy of the predictions for each individual power vector component was substantially improved resulting in a ± 0.63 D, ±0.14D, and ±0.08 D reduction in the 95% limits of agreement of the error distribution for M, J0, and J45, respectively. The wavefront-aberrometry related variables had the biggest impact on the prediction, while demographic and measurement quality-related features showed a heterogeneous but consistent predictive value. CONCLUSIONS: These results suggest that ML is effective for improving precision in predicting patient's SR from objective measurements taken with a low-cost portable device.

Predicting subjective refraction with dynamic retinal image quality analysis.

The aim of this work is to evaluate the performance of a novel algorithm that combines dynamic wavefront aberrometry data and descriptors of the retinal image quality from objective autorefractor measurements to predict subjective refraction. We conducted a retrospective study of the prediction accuracy and precision of the novel algorithm compared to standard search-based retinal image quality optimization algorithms. Dynamic measurements from 34 adult patients were taken with a handheld wavefront autorefractor and static data was obtained with a high-end desktop wavefront aberrometer. The search-based algorithms did not significantly improve the results of the desktop system, while the dynamic approach was able to simultaneously reduce the standard deviation (up to a 15% for reduction of spherical equivalent power) and the mean bias error of the predictions (up to 80% reduction of spherical equivalent power) for the handheld aberrometer. These results suggest that dynamic retinal image analysis can substantially improve the accuracy and precision of the portable wavefront autorefractor relative to subjective refraction.

Investigation of the Accuracy of a Low-Cost, Portable Autorefractor to Provide Well-Tolerated Eyeglass Prescriptions: A Randomized Crossover Trial.

PURPOSE: To compare patient preferences for eyeglasses prescribed using a low-cost, portable wavefront autorefractor versus standard subjective refraction (SR). DESIGN: Randomized, cross-over clinical trial. PARTICIPANTS: Patients aged 18 to 40 years presenting with refractive errors (REs) to a tertiary eye hospital in Southern India. METHODS: Participants underwent SR followed by autorefraction (AR) using the monocular version of the QuickSee device (PlenOptika Inc). An independent optician, masked to the refraction approach, prepared eyeglasses based on each refraction approach. Participants (masked to refraction source) were randomly assigned to use SR- or AR-based eyeglasses first, followed by the other pair, for 1 week each. At the end of each week, participants had their vision checked and were interviewed about their experience with the eyeglasses. MAIN OUTCOME MEASURES: Patients preferring eyeglasses were chosen using AR and SR. RESULTS: The 400 participants enrolled between March 26, 2018, and August 2, 2019, had a mean (standard deviation) age of 28.4 (6.6) years, and 68.8% were women. There was a strong correlation between spherical equivalents using SR and AR (r = 0.97, P < 0.001) with a mean difference of -0.07 diopters (D) (95% limits of agreement [LoA], -0.68 to 0.83). Of the 301 patients (75.2%) who completed both follow-up visits, 50.5% (n = 152) and 49.5% (n = 149) preferred glasses prescribed using SR and AR, respectively (95% CI, 45.7-56.3; P = 0.86). There were no differences in demographic or vision characteristics between participants with different preferences (P > 0.05 for all). CONCLUSIONS: We observed a strong agreement between the prescriptions from SR and AR, and eyeglasses prescribed using SR and AR were equally preferred by patients. Wider use of prescribing based on AR alone in resource-limited settings is supported by these findings.

Coding and non-coding roles of MOCCI (C15ORF48) coordinate to regulate host inflammation and immunity.

Mito-SEPs are small open reading frame-encoded peptides that localize to the mitochondria to regulate metabolism. Motivated by an intriguing negative association between mito-SEPs and inflammation, here we screen for mito-SEPs that modify inflammatory outcomes and report a mito-SEP named "Modulator of cytochrome C oxidase during Inflammation" (MOCCI) that is upregulated during inflammation and infection to promote host-protective resolution. MOCCI, a paralog of the NDUFA4 subunit of cytochrome C oxidase (Complex IV), replaces NDUFA4 in Complex IV during inflammation to lower mitochondrial membrane potential and reduce ROS production, leading to cyto-protection and dampened immune response. The MOCCI transcript also generates miR-147b, which targets the NDUFA4 mRNA with similar immune dampening effects as MOCCI, but simultaneously enhances RIG-I/MDA-5-mediated viral immunity. Our work uncovers a dual-component pleiotropic regulation of host inflammation and immunity by MOCCI (C15ORF48) for safeguarding the host during infection and inflammation.

Assesment of the QuickSee wavefront autorefractor for characterizing refractive errors in school-age children.

PURPOSE: To assess the performance of an open-view binocular handheld aberrometer (QuickSee) for diagnosing refractive errors in children. METHODS: 123 school-age children (9.9 ± 3.3 years) with moderate refractive error underwent autorefraction (AR) with a standard desktop device and subjective refraction (SR), with or without cycloplegia to determine their eyeglass prescription. Measurements with QuickSee (QS) were taken in 62 of these patients without cycloplegia (NC), and in 61 under cycloplegia (C). Differences in refraction values (AR vs SR vs QS) as well as the visual acuity (VA) achieved by the patients with each method (QS vs SR) were used to evaluate the performance of the device in measuring refractive error. RESULTS: The spherical equivalent refraction obtained by QS agreed within 0.5 D of the SR in 71% (NC) and 70% (C) of the cases. Agreement between the desktop autorefractor and SR for the same threshold was of 61% (NC) and 77% (C). VA resulting from QS refractions was equal to or better than that achieved by SR procedure in 77% (NC) and 74% (C) of the patients. Average improvement in VA with the QS refractions was of 8.6 and 13.4 optotypes for the NC and C groups respectively, while the SR procedure provided average improvements of 8.9 (NC) and 14.8 (C) optotypes. CONCLUSIONS: The high level of agreement between QuickSee and subjective refraction together with the VA improvement achieved in both study groups using QuickSee refractions suggest that the device is a useful autorefraction tool for school-age children.

Validation of an Affordable Handheld Wavefront Autorefractor.

SIGNIFICANCE: There is a critical need for tools that increase the accessibility of eye care to address the most common cause of vision impairment: uncorrected refractive errors. This work assesses the performance of an affordable autorefractor, which could help reduce the burden of this health care problem in low-resource communities. PURPOSE: The purpose of this study was to validate the commercial version of a portable wavefront autorefractor for measuring refractive errors. METHODS: Refraction was performed without cycloplegia using (1) a standard clinical procedure consisting of an objective measurement with a desktop autorefractor followed by subjective refraction (SR) and (2) with the handheld autorefractor. Agreement between both methods was evaluated using Bland-Altman analysis and by comparing the visual acuity (VA) with trial frames set to the resulting measurements. RESULTS: The study was conducted on 54 patients (33.9 ± 14.1 years of age) with a spherical equivalent (M) refraction determined by SR ranging from -7.25 to 4.25 D (mean ± SD, -0.93 ± 1.95 D). Mean differences between the portable autorefractor and SR were 0.09 ± 0.39, -0.06 ± 0.13, and 0.02 ± 0.12 D for M, J0, and J45, respectively. The device agreed within 0.5 D of SR in 87% of the eyes for spherical equivalent power. The average VAs achieved from trial lenses set to the wavefront autorefractor and SR results were 0.02 ± 0.015 and 0.015 ± 0.042 logMAR units, respectively. Visual acuity resulting from correction based on the device was the same as or better than that achieved by SR in 87% of the eyes. CONCLUSIONS: This study found excellent agreement between the measurements obtained with the portable autorefractor and the prescriptions based on SR and only small differences between the VA achieved by either method.

Quality of eyeglass prescriptions from a low-cost wavefront autorefractor evaluated in rural India: results of a 708-participant field study.

OBJECTIVE: To assess the quality of eyeglass prescriptions provided by an affordable wavefront autorefractor operated by a minimally trained technician in a low-resource setting. METHODS AND ANALYSIS: 708 participants were recruited from consecutive patients registered for routine eye examinations at Aravind Eye Hospital in Madurai, India, or an affiliated rural satellite vision centre. Visual acuity (VA) and patient preference were compared between trial lenses set to two eyeglass prescriptions from (1) a novel wavefront autorefractor and (2) subjective refraction by an experienced refractionist. RESULTS: The mean±SD VA was 0.30±0.37, -0.02±0.14 and -0.04±0.11 logarithm of the minimum angle of resolution units before correction, with autorefractor correction and with subjective refraction correction, respectively (all differences p<0.01). Overall, 25% of participants had no preference, 33% preferred eyeglass prescriptions from autorefraction, and 42% preferred eyeglass prescriptions from subjective refraction (p<0.01). Of the 438 patients 40 years old and younger, 96 had no preference and the remainder had no statistically significant difference in preference for subjective refraction prescriptions (51%) versus autorefractor prescriptions (49%) (p=0.52). CONCLUSION: Average VAs from autorefractor-prescribed eyeglasses were one letter worse than those from subjective refraction. More than half of all participants either had no preference or preferred eyeglasses prescribed by the autorefractor. This marginal difference in quality may warrant autorefractor-based prescriptions, given the portable form factor, short measurement time, low cost and minimal training required to use the autorefractor evaluated here.

Design and Clinical Evaluation of a Handheld Wavefront Autorefractor.

PURPOSE: To introduce a novel autorefractor design that is intended to be manufacturable at low cost and evaluate its performance in measuring refractive errors. METHODS: We developed a handheld, open-view autorefractor (the "QuickSee" [QS]) that uses a simplified approach to wavefront sensing that forgoes moving parts and expensive components. Adult subjects (n = 41) were recruited to undergo noncycloplegic refraction with three methods: (1) a QS prototype, (2) a Grand Seiko WR-5100K (GS) autorefractor, and (3) subjective refraction (SR). Agreements between the QS and GS were evaluated using a Bland-Altman analysis. The accuracy of both autorefractors was evaluated using SR as the clinical gold standard. RESULTS: The spherical equivalent powers measured from both autorefractors correlate well with SR, with identical correlation coefficients of r = 0.97. Both autorefractors also agree well with each other, with a spherical equivalent power 95% confidence interval of ±0.84 diopters (D). The difference between the accuracy of each objective device is not statistically significant for any component of the power vector (p = 0.55, 0.41, and 0.18, for M, J0, and J45, respectively). The spherical and cylindrical powers measured by the GS agree within 0.25 D of the SR in 49 and 82% of the eyes, respectively, whereas the spherical and cylindrical powers measured by the QS agree within 0.25 D of the SR in 74 and 87% of the eyes, respectively. CONCLUSIONS: The prototype autorefractor exhibits equivalent performance to the GS autorefractor in matching power vectors measured by SR.

From unseen to seen: tackling the global burden of uncorrected refractive errors.

Worldwide, more than one billion people suffer from poor vision because they do not have the eyeglasses they need. Their uncorrected refractive errors are a major cause of global disability and drastically reduce productivity, educational opportunities, and overall quality of life. The problem persists most prevalently in low-resource settings, even though prescription eyeglasses serve as a simple, effective, and largely affordable solution. In this review, we discuss barriers to obtaining, and approaches for providing, refractive eye care. We also highlight emerging technologies that are being developed to increase the accessibility of eye care. Finally, we describe opportunities that exist for engineers to develop new solutions to positively impact the diagnosis and treatment of correctable refractive errors in low-resource settings.

Photometric stereo endoscopy.

While color video endoscopy has enabled wide-field examination of the gastrointestinal tract, it often misses or incorrectly classifies lesions. Many of these missed lesions exhibit characteristic three-dimensional surface topographies. An endoscopic system that adds topographical measurements to conventional color imagery could therefore increase lesion detection and improve classification accuracy. We introduce photometric stereo endoscopy (PSE), a technique which allows high spatial frequency components of surface topography to be acquired simultaneously with conventional two-dimensional color imagery. We implement this technique in an endoscopic form factor and demonstrate that it can acquire the topography of small features with complex geometries and heterogeneous optical properties. PSE imaging of ex vivo human gastrointestinal tissue shows that surface topography measurements enable differentiation of abnormal shapes from surrounding normal tissue. Together, these results confirm that the topographical measurements can be obtained with relatively simple hardware in an endoscopic form factor, and suggest the potential of PSE to improve lesion detection and classification in gastrointestinal imaging.

Fast optically sectioned fluorescence HiLo endomicroscopy.

We describe a nonscanning, fiber bundle endomicroscope that performs optically sectioned fluorescence imaging with fast frame rates and real-time processing. Our sectioning technique is based on HiLo imaging, wherein two widefield images are acquired under uniform and structured illumination and numerically processed to reject out-of-focus background. This work is an improvement upon an earlier demonstration of widefield optical sectioning through a flexible fiber bundle. The improved device features lateral and axial resolutions of 2.6 and 17 µm, respectively, a net frame rate of 9.5 Hz obtained by real-time image processing with a graphics processing unit (GPU) and significantly reduced motion artifacts obtained by the use of a double-shutter camera. We demonstrate the performance of our system with optically sectioned images and videos of a fluorescently labeled chorioallantoic membrane (CAM) in the developing G. gallus embryo. HiLo endomicroscopy is a candidate technique for low-cost, high-speed clinical optical biopsies.

Optically sectioned in vivo imaging with speckle illumination HiLo microscopy.

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Practical implementation of log-scale active illumination microscopy.

Active illumination microscopy (AIM) is a method of redistributing dynamic range in a scanning microscope using real-time feedback to control illumination power on a sub-pixel time scale. We describe and demonstrate a fully integrated instrument that performs both feedback and image reconstruction. The image is reconstructed on a logarithmic scale to accommodate the dynamic range benefits of AIM in a single output channel. A theoretical and computational analysis of the influence of noise on active illumination feedback is presented, along with imaging examples illustrating the benefits of AIM. While AIM is applicable to any type of scanning microscope, we apply it here specifically to two-photon microscopy.

Widefield fluorescence sectioning with HiLo microscopy.

HiLo microscopy is a widefield fluorescence imaging technique that provides depth discrimination by combining two images, one with non-uniform illumination and one with uniform illumination. We discuss the theory of this technique and a variety of practical implementations in brain-tissue imaging and fluorescence endomicroscopy.

Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle.

We present an endomicroscope apparatus that exhibits out-of-focus background rejection based on wide-field illumination through a flexible imaging fiber bundle. Our technique, called HiLo microscopy, involves acquiring two images, one with grid-pattern illumination and another with standard uniform illumination. An evaluation of the image contrast with grid-pattern illumination provides an optically sectioned image with low resolution. This is complemented with high-resolution information from the uniform illumination image, leading to a full-resolution image that is optically sectioned. HiLo endomicroscope movies are presented of fluorescently labeled rat colonic mucosa.

Stochastic actin polymerization and steady retrograde flow determine growth cone advancement.

Neuronal growth is an extremely complex yet reliable process that is directed by a dynamic lamellipodial structure at the tip of every growing neurite, called the growth cone. Lamellipodial edge fluctuations are controlled by the interplay between actin polymerization pushing the edge forward and molecular motor driven retrograde actin flow retracting the actin network. The leading edge switches randomly between extension and retraction processes. We identify switching of "on/off" states in actin polymerization as the main determinant of lamellipodial advancement. Our analysis of motility statistics allows for a prediction of growth direction. This was used in simulations explaining the amazing signal detection capabilities of neuronal growth by the experimentally found biased stochastic processes. Our measurements show that the intensity of stochastic fluctuations depend on changes in the underlying active intracellular processes and we find a power law eta = a*x(alpha) with exponent alpha = 2.63 +/- 0.12 between noise intensity eta and growth cone activity x, defined as the sum of protrusion and retraction velocity. Differences in the lamellipodial dynamics between primary neurons and a neuronal cell line further suggests that active processes tune the observed stochastic fluctuations. This hints at a possible role of noise intensity in determining signal detection sensitivity.