Membrane Protein Detection and Morphological Analysis of Red Blood Cells in Hereditary Spherocytosis by Confocal Laser Scanning Microscopy.

In hereditary spherocytosis (HS), genetic mutations in the cell membrane and cytoskeleton proteins cause structural defects in red blood cells (RBCs). As a result, cells are rigid and misshapen, usually with a characteristic spherical form (spherocytes), too stiff to circulate through microcirculation regions, so they are prone to undergo hemolysis and phagocytosis by splenic macrophages. Mild to severe anemia arises in HS, and other derived symptoms like splenomegaly, jaundice, and cholelithiasis. Although abnormally shaped RBCs can be identified under conventional light microscopy, HS diagnosis relies on several clinical factors and sometimes on the results of complex molecular testing. It is specially challenging when other causes of anemia coexist or after recent blood transfusions. We propose two different approaches to characterize RBCs in HS: (i) an immunofluorescence assay targeting protein band 3, which is affected in most HS cases and (ii) a three-dimensional morphology assay, with living cells, staining the membrane with fluorescent dyes. Confocal laser scanning microscopy (CLSM) was used to carry out both assays, and in order to complement the latter, a software was developed for the automated detection of spherocytes in blood samples. CLSM allowed the precise and unambiguous assessment of cell shape and protein expression.

Optical Technologies for the Improvement of Skin Cancer Diagnosis: A Review.

The worldwide incidence of skin cancer has risen rapidly in the last decades, becoming one in three cancers nowadays. Currently, a person has a 4% chance of developing melanoma, the most aggressive form of skin cancer, which causes the greatest number of deaths. In the context of increasing incidence and mortality, skin cancer bears a heavy health and economic burden. Nevertheless, the 5-year survival rate for people with skin cancer significantly improves if the disease is detected and treated early. Accordingly, large research efforts have been devoted to achieve early detection and better understanding of the disease, with the aim of reversing the progressive trend of rising incidence and mortality, especially regarding melanoma. This paper reviews a variety of the optical modalities that have been used in the last years in order to improve non-invasive diagnosis of skin cancer, including confocal microscopy, multispectral imaging, three-dimensional topography, optical coherence tomography, polarimetry, self-mixing interferometry, and machine learning algorithms. The basics of each of these technologies together with the most relevant achievements obtained are described, as well as some of the obstacles still to be resolved and milestones to be met.

Spectroscopic Evaluation of Red Blood Cells of Thalassemia Patients with Confocal Microscopy: A Pilot Study.

Hemoglobinopathies represent the most common single-gene defects in the world and pose a major public health problem, particularly in tropical countries, where they occur with high frequency. Diagnosing hemoglobinopathies can sometimes be difficult due to the coexistence of different causes of anemia, such as thalassemia and iron deficiency, and blood transfusions, among other factors, and requires expensive and complex molecular tests. This work explores the possibility of using spectral confocal microscopy as a diagnostic tool for thalassemia in pediatric patients, a disease caused by mutations in the globin genes that result in changes of the globin chains that form hemoglobin-in pediatric patients. Red blood cells (RBCs) from patients with different syndromes of alpha-thalassemia and iron deficiency (including anemia) as well as healthy (control) subjects were analyzed under a Leica TCS SP8 confocal microscope following different image acquisition protocols. We found that diseased RBCs exhibited autofluorescence when excited at 405 nm and their emission was collected in the spectral range from 425 nm to 790 nm. Three experimental descriptors calculated from the mean emission intensities at 502 nm, 579 nm, 628 nm, and 649 nm allowed us to discriminate between diseased and healthy cells. According to the results obtained, spectral confocal microscopy could serve as a tool in the diagnosis of thalassemia.

Experimental characterization of the speckle pattern at the output of a multimode optical fiber.

Speckle patterns produced by coherent waves interfering with each other are undesirable in many imaging applications (for example, in laser projection systems) but on the other hand, they contain useful information that can be exploited (for example, for blood flow analysis or reconstruction of the object that generates the speckle). It is therefore important to understand how speckle can be enhanced or reduced by tailoring the coherence of laser light. Using a conventional semiconductor laser and a multimode optical fiber we study experimentally how the speckle pattern depends on the laser pump current and on the image acquisition settings. By varying the pump current from below to above the lasing threshold, and simultaneously tuning the image exposure time to compensate for the change in brightness, we find conditions that allow for recorded images with similar average intensity, but with speckle contrast (the standard deviation of the intensity over the average intensity) as low as 0.16, or as high as 0.99.

Method to reduce undesired multiple fundus scattering effects in double-pass systems.

Double-pass systems rely on backscattering of light by the human ocular fundus to assess the optical quality of the eye. In this work, we present a method to reduce double-pass image degradation caused by undesired multiple scattering effects in the eye fundus. The reduction is based on combined data processing of simultaneous measurements using two different configurations: one symmetric with equal entrance and exit pupils and another asymmetric with unequal entrance and exit pupils. Under certain conditions, such scattering effects may be effectively suppressed. Measurements of human eyes show that, although multiple fundus scattering imposes a shift on the estimations, double-pass systems can be used to predict the optical quality of the eye within a population.

Visible and Extended Near-Infrared Multispectral Imaging for Skin Cancer Diagnosis.

With the goal of diagnosing skin cancer in an early and noninvasive way, an extended near infrared multispectral imaging system based on an InGaAs sensor with sensitivity from 995 nm to 1613 nm was built to evaluate deeper skin layers thanks to the higher penetration of photons at these wavelengths. The outcomes of this device were combined with those of a previously developed multispectral system that works in the visible and near infrared range (414 nm⁻995 nm). Both provide spectral and spatial information from skin lesions. A classification method to discriminate between melanomas and nevi was developed based on the analysis of first-order statistics descriptors, principal component analysis, and support vector machine tools. The system provided a sensitivity of 78.6% and a specificity of 84.6%, the latter one being improved with respect to that offered by silicon sensors.

Validation of a gonio-hyperspectral imaging system based on light-emitting diodes for the spectral and colorimetric analysis of automotive coatings.

In this study, a novel gonio-hyperspectral imaging system based on light-emitting diodes for the analysis of automotive coatings was validated colorimetrically and spectrally from 368 to 1309 nm. A total of 30 pearlescent, 30 metallic, and 30 solid real automotive coatings were evaluated with this system, the BYK-mac and X-Rite MA98 gonio-spectrophotometers, and also with the SPECTRO 320 spectrometer for further comparison. The results showed very precise correlations, especially in the visible range. In conclusion, this new system provides a deeper assessment of goniochromatic pigments than current approaches due to the expansion of the spectral range to the infrared.

Repeatability of aberrometric measurements with a new instrument for vision analysis based on adaptive optics.

PURPOSE: To evaluate intersession and intrasession repeatability of aberration data obtained with a new visual simulator based on adaptive optics, which includes a Hartmann-Shack aberrometer (Adaptive Optics Vision Analyzer; Voptica S.L., Murcia, Spain). METHODS: Thirty-one healthy right eyes were included in the study. To evaluate intrasession repeatability, three consecutive measurements without repositioning the patient or realigning the eye were obtained. Intersession repeatability was evaluated in three sessions. Aberrometric data computed from the second to the fifth order for a 4-mm pupil were used. Statistical analysis included the repeated measures analysis of variance (or the Wilcoxon signed rank test), the coefficient of repeatability, the Bland-Altman method, and the intraclass correlation coefficient. RESULTS: No significant differences in the intrasession and intersession repeatability analysis for any of the parameters (P > .05) were found, suggesting a consistent variability of the instrument over time. Similar coefficient of repeatability values were obtained in the three sessions. The Bland-Altman analysis confirmed differences close to zero and the variations were independent of the mean within and between sessions. The intersession intraclass correlation coefficient values were generally above 0.75, suggesting moderate to high repeatability. However, some exceptions were found in the intrasession analysis. CONCLUSIONS: The findings suggest that the new instrument provides consistent and repeatable aberrometric data. It is therefore a suitable tool to perform consistent and repeatable visual simulations.

Comparing Autorefractors for Measurement of Accommodation.

PURPOSE: To compare the static and dynamic accommodative responses measured with the WAM-5500 and the PowerRef-II autorefractors. METHODS: The dynamic and static monocular accommodative responses were measured with the WAM-5500 and the PowerRef-II instruments in 30 pre-presbyopic patients (23.66 [±3.19] years). The spherical equivalent was measured at 0.00, 2.50, and 5.00 diopters (D) of accommodative stimulation for the static measurements. The subjective refraction was also determined. Dynamic accommodation was measured for abrupt changes of stimulus vergence of 2.00 D. Mean and peak velocities of accommodation and disaccommodation were evaluated. For the PowerRef-II, dynamic measurements were calculated for sampling frequencies of 5 and 25 Hz. RESULTS: For far distance static results, the differences between subjective and WAM-5500 measurements were 0.07 (±0.21) D (p = 0.093) and those between subjective and PowerRef-II measurements were 0.70 (±0.47) D (p = 0.001). The difference in the response measured with both instruments was 0.08 (±0.32) D (p = 0.194) for 2.50 D and -0.32 (±0.48) D (p = 0.001) for 5.00 D of stimulation. For the dynamic mode, the PowerRef-II at 25 Hz measured faster mean and peak velocities of accommodation and disaccommodation than the WAM-5500, with statistically significant (p < 0.05) differences of 0.68 (±1.01), 0.67 (±0.98), 1.26 (±1.19), and 1.42 (±1.53) D/s, respectively. With a sampling frequency of 5 Hz for the PowerRef-II, these differences, which were statistically significant (p < 0.05), were reduced to 0.52 (±0.90), 0.49 (±0.91), 0.83 (±1.07), and 0.83 (±1.31) D/s, respectively. CONCLUSIONS: There is good agreement between subjective refraction and WAM-5500 measurements. In contrast, the PowerRef-II produced more hyperopic results. There were no differences among instruments at 2.50 D of static stimulation; however, differences were found at 5.00 D. In the dynamic measurements, the PowerRef-II measured faster velocities, partly attributed to the difference in the sampling frequency.

Portable multispectral imaging system based on light-emitting diodes for spectral recovery from 370 to 1630 nm.

To expand and investigate the potential of spectral imaging, we developed a portable multispectral system using light-emitting diodes. This system recovers spectral information from the UV to the near IR over a large area using two different image sensors synchronized with 23 bands of illumination. The system was assessed for spectral reconstruction through simulations and experimental measurements by means of two methods of spectral reconstruction and three different evaluation metrics. The results over a Macbeth ColorChecker chart and other samples, including pigments usually employed in art paintings, are compared and discussed. The portable multispectral system using LEDs constitutes a cost-effective and versatile method for spectral imaging.

Age-related changes in accommodation measured with a double-pass system.

PURPOSE: To measure the accommodative response in a wide age range population using the retinal image quality assessments provided by a double-pass system. METHODS: The accommodative response was measured using a custom-built double-pass setup in 84 patients from 15 to 55 years of age. Patients were classified in four groups (A: 15-25 year old; B: 26-35; C: 36-45; D: 46-55). Accommodation was stimulated from 0 to 5 dioptres (D) with the push up method using an open field fixation test. The total accommodative response in the stimulated range, the accommodative stimulus-response curve, the slope and the progression of optical quality with accommodation were measured. RESULTS: The total accommodation obtained in groups A and B was high, with a mean value of 4 D or higher, whereas values in older individuals were significantly lower. The accommodative stimulus-response curve and the slope were also high with a sudden decrease in patients over 35 years of age. The optical quality of the eye during accommodation did not change significantly. CONCLUSIONS: Participants aged 15-35 years showed a good accommodative response. Thereafter, a significant decrease of the response in the total accommodation, stimulus response curve and slope was observed. The younger groups showed a larger accommodative response than previously published.

Influence of power and the time of application of fogging lenses on accommodation.

Fogging is a non-invasive technique based on the use of positive spherical power lenses to relax accommodation during refraction that is commonly used as an alternative to cycloplegic drugs. Although the mechanism of the fogging technique has been described, some aspects of its methodology remain unclear. The main purpose of this work was to determine which lens power and time of application are more suitable for achieving a successful relaxation of accommodation by analyzing the changes in accommodation when fogging lenses of different powers were placed in front of the participants' eye for a certain timespan. The results of this analysis showed, in general, that low-power lenses and timespans of less than half a minute provided the highest relaxation of accommodation. However, high inter-subject variability was found in the two variables (power and time).

System for Objective Assessment of the Accommodation Response During Subjective Refraction.

Purpose: To evaluate a system based on a Hartmann-Shack wavefront sensor attached to a phoropter that allows the user to obtain real-time information about the refractive state of the eye and the accommodation response (AR). Methods: The objective refractions (ME) and ARs of 73 subjects (50 women, 23 men; ages, 19-69 years) were assessed with the system developed while placing in the phoropter the subjective refraction (MS) plus a set of trial lenses with differences in spherical equivalent power (ΔM) between ±2 diopters (D). Results: The objective estimations (ME) showed a good correlation with the subjective values (MS) (r = 0.989; P < 0.001). The means of the ARs presented a region where the accommodation remained stable (ΔM from +2 D to about 0 D), followed by another in which the response increased progressively (ΔM from about 0 to -2 D) with the magnitude of the accommodation stimulus. The analysis of variance within subjects applied to ARs introducing age and MS as covariates showed an increasing effect size of age from medium to large between ΔM of -0.5 and -2 D. In contrast, MS had a medium effect size (between ΔM of +2 and 0 D). Conclusions: The implemented system permitted an objective estimation of the refraction of the eye and its AR. Because it is coupled to a phoropter, the system can be used to retrieve the AR during subjective refraction procedures. Translational Relevance: The developed system can be used as a supporting tool during subjective refraction to provide certainty about the true state of accommodation.

Eye movements in patients with post-COVID condition.

Eye movement control is impaired in some neurological conditions, but the impact of COVID-19 on eye movements remains unknown. This study aims to investigate differences in oculomotor function and pupil response in individuals who suffer post-COVID-19 condition (PCC) with cognitive deficits. Saccades, smooth pursuit, fixation, vergence and pupillary response were recorded using an eye tracker. Eye movements and pupil response parameters were computed. Data from 16 controls, 38 COVID mild (home recovery) and 19 COVID severe (hospital admission) participants were analyzed. Saccadic latencies were shorter in controls (183 ± 54 ms) than in COVID mild (236 ± 83 ms) and COVID severe (227 ± 42 ms) participants (p = 0.017). Fixation stability was poorer in COVID mild participants (Bivariate Contour Ellipse Area of 0.80 ± 1.61°2 vs 0.36 ± 0.65 °2 for controls, p = 0.019), while percentage of pupil area reduction/enlargement was reduced in COVID severe participants (39.7 ± 12.7%/31.6 ± 12.7% compared to 51.7 ± 22.0%/49.1 ± 20.7% in controls, p < 0.015). The characteristics of oculomotor alterations found in PCC may be useful to understand different pathophysiologic mechanisms.

Comparison between an objective and a psychophysical method for the evaluation of intraocular light scattering.

In this study we present the comparison of the performance of two systems to measure intraocular scattering. Measurements were made by using a psychophysical system based on a brightness comparison method that provides a glare index and a physical system based on the double-pass technique, which gives an objective scatter index by measuring the optical quality of the eye. Three external diffuser filters that simulated different grades of intraocular scattering were used in subjects with normal vision. The two measured indexes showed a graded rise with increasing level of scattering. The discrimination ability obtained for both systems showed that they were able to distinguish among conditions ranging from normal to early cataracts.

Reflectance evaluation of eye fundus structures with a visible and near-infrared multispectral camera.

We examined the spectral reflectance of fundus structures in the visible and near-infrared (400-1300 nm) range for contributing to the medical diagnosis of fundus diseases. Spectral images of healthy eye fundus and other ocular diseases were acquired using a novel multispectral fundus camera. Reflectance metrics were computed based on contrast to analyze the spectral features. Significant differences were observed among the structures in healthy and diseased eye fundus. Specifically, near-infrared analysis allows imaging of deeper layers, such as the choroid, which, to date, has not been retrieved using traditional color fundus cameras. Pathological structures, which were hardly observable in color fundus images owing to metamerism, were also revealed by the developed multispectral fundus camera.

Intra- and intersession repeatability of a double-pass instrument.

PURPOSE: To evaluate the intra- and intersession repeatability of the optical quality parameters provided by the Optical Quality Analysis System (OQAS), which is based on the double-pass technique. METHODS: We performed optical quality measurements using the OQAS on 20 eyes of 10 healthy subjects who had best spectacle-corrected visual acuity of 20/20 or better. Measurements were performed by the same examiner in three different sessions that were separated by 10-min intervals. The subject's eye was realigned at the beginning of each session. During each session, three consecutive measurements were taken without realignment. The following optical quality parameters were analyzed: the modulation transfer function cutoff frequency (MTFcutoff), the Strehl ratio, the OQAS values (OV) at contrasts of 100%, 20%, and 9%, and the objective scatter index (OSI). RESULTS: The mean coefficients of repeatability obtained for the first session were 4.51 (MTFcutoff), 0.049 (Strehl ratio), 0.15 (OV 100%), 0.21 (OV 20%), 0.28 (OV 9%), and 0.11 (OSI), which were similar to those found in the second and third sessions. The confidence limits in the Bland and Altman charts when the intrasession repeatability was assessed (in a comparison of the first and second measurements of the first session) ranged from -3.16 to 3.94 (MTFcutoff), -0.060 to 0.069 (Strehl ratio), -0.12 to 0.18 (OV 100%), -0.20 to 0.23 (OV 20%), -0.29 to 0.27 (OV 9%), and -0.12 to 0.13 (OSI). The same limits when the intersession repeatability was assessed (in a comparison of the first and second sessions) ranged from -5.30 to 5.49 (MTFcutoff), -0.054 to 0.050 (Strehl ratio), -0.17 to 0.17 (OV 100%), -0.22 to 0.19 (OV 20%), -0.26 to 0.29 (OV 9%), and -0.12 to 0.13 (OSI). CONCLUSIONS: Our findings showed that OQAS is a clinical instrument with a good intra- and intersession repeatability and that the realignment of the eye does not introduce any additional variability in the measurements.

Temporal stability in the perception of dry eye ocular discomfort symptoms.

PURPOSE: A prospective longitudinal study was designed to investigate the ability of patients with tear deficiency to correctly recall their past symptoms. The ultimate goal of the study was to contribute to the ongoing research concerning the lack of association between dry eye symptomatology and clinical tests of tear film evaluation. METHODS: A total of 26 subjects with ages ranging from 29 to 61 years participated in the study. All subjects reported symptoms associated with tear deficiency, although none had been diagnosed with dry eye disease. Subjects were instructed to grade their symptoms on two different occasions, at the precise moment they were experiencing them, by means of a home questionnaire, and through a recall questionnaire, which was administered within a maximum interval of 10 days from the first questionnaire. Tear evaluation tests were performed at this second time. Non-parametric statistical analyses were used to investigate the relationship between present and recalled symptoms and between symptoms and signs, as well as between the different dry eye tests. The contributions of age, gender, and recall period were also evaluated. RESULTS: With the exception of irritation (p = 0.029) and scratchiness (p = 0.025), no statistically significant difference was encountered between home and recall questionnaires, although females were found to recall their symptoms slightly better than males (p = 0.048). An increase in the severity of the symptoms was associated with a better recollection (p = 0.007). Symptoms (home or recalled) and clinical signs were not correlated, although the recalled symptom of scratchiness presented moderately strong correlations with several dry eye tests. CONCLUSIONS: Although the lack of correlation between dry eye tests and symptoms mirrored previous research, symptoms recall was found to follow certain interesting patterns, similar to those published in pain research literature.

Optical quality one month after verisyse and Veriflex phakic IOL implantation and Zeiss MEL 80 LASIK for myopia from 5.00 to 16.50 diopters.

PURPOSE: To evaluate the eye's optical quality after phakic intraocular lens (IOL) implantation and LASIK for moderate to high myopia. METHODS: The retinal image quality of 45 patients was evaluated after undergoing one of three surgical procedures (9 patients with Verisyse IOL [AMO] implants, 11 patients with Veriflex IOL [AMO] implants, and 25 patients who underwent LASIK). Patients were aged <40 years, had at least 5.00 diopters of myopia, and had preoperative best spectacle-corrected visual acuity and postoperative uncorrected visual acuity better than 20/30. The eye's optical quality was measured using the Optical Quality Analysis System (OQAS, Visiometrics S.L.), which is an instrument based on the double-pass technique. Measurements were performed before surgery and 1 day and 1 month after surgery. RESULTS: Optical quality worsened noticeably 1 day after surgery with the Verisyse IOL with a 50% to 60% loss, most likely due to the large incision and the presence of sutures in most eyes. The LASIK technique and Veriflex IOL implant did not cause as remarkable a decrease in optical quality (20% to 25% loss). One month after surgery, the optical quality of patients with IOL implants was high, although some surgically induced astigmatism remained, especially in the Verisyse patients. Conversely, LASIK patients had slightly lower optical quality, with optical parameters that represented 90% of their initial value. CONCLUSIONS: Verisyse and Veriflex phakic IOL implantation and LASIK are both safe and effective in correcting moderate to high myopia, but they involve different processes of optical quality recovery. One day after surgery, the Verisyse IOL implantation significantly reduced the eye's optical quality, mainly due to the larger incision required and the higher number of sutures used. This reduction was not as remarkable with the other two techniques. However, 1 month after surgery, patients with IOL implants recovered more optical quality than LASIK patients.

Speckle reduction in double-pass retinal images.

The double pass (DP) technique quantifies the optical quality of the eye by measuring its point spread function. The low reflectivity of the retina requires the use of a high-brightness, point-like illumination source, and thus, DP systems use laser diodes (LDs). However, LDs light produces speckle, and a low-cost solution to reduce speckle is to include a vibrating mirror in the beam path. With the goal of finding an all-optical solution, here we perform a comparative study of the amount of speckle produced by three semiconductor light sources: an LD, a light emitting diode (LED), and a superluminescent diode (SLED). We also compare the results with the speckle reduction that is obtained with a vibrating mirror. We find that the SLED is a good alternative to LD illumination, as the amount of speckle in the image is almost as low as that obtained with an LD and a vibrating mirror in the beam path.