In vivo assessment of human corneal epithelial cells in orthokeratology lens wearers: A pilot study.

SIGNIFICANCE: Central corneal epithelial thinning associated with midperipheral epithelial thickening has been reported as the main factor contributing to the effectiveness of orthokeratology (ortho-k) in myopia control. Yet, the cellular mechanism governing the regional change in refractive power remains elusive. PURPOSE: This study aimed to evaluate the correlation between the regional change in corneal epithelial thickness and cell density in ortho-k wearers. METHODS: A new human prototype of a polarization-dependent optical coherence microscope was developed to enable noncontact and noninvasive in vivo imaging of corneal epithelial cells in ortho-k wearers with and without their ortho-k lens. The epithelial thickness and cell density were evaluated at the central and midperipheral corneal locations in four ortho-k wearers and four spectacle wearers serving as controls. RESULTS: Polarization-dependent optical coherence microscope achieved in vivo volumetric imaging of all epithelial cell types in ortho-k wearers with and without their lens over a field of view of 0.5 × 0.5 mm 2 with an isotropic resolution of ~2.2 mm. The central epithelial thinning and midperipheral epithelial thickening were consistent across all ortho-k wearers. However, the inconsistency in their regional epithelial cell density highlighted a great variability in individual response to ortho-k treatment. There was no strong correlation between epithelial thickness and cell density, especially at the midperipheral cornea, in ortho-k participants. CONCLUSIONS: This study constitutes our first step toward uncovering the cellular mechanism underlying the effectiveness of ortho-k in myopia control. Future studies will focus on the longitudinal evaluation of epithelial cells before and during ortho-k treatment to identify factors governing individual response to ortho-k treatment and ultimately inform the dynamics of epithelial cells taking place during the ortho-k treatment.

Non-invasive in vivo imaging of human corneal microstructures with optical coherence microscopy.

Non-invasive imaging systems with cellular-level resolution offer the opportunity to identify biomarkers of the early stage of corneal diseases, enabling early intervention, monitoring of disease progression, and evaluating treatment efficacy. In this study, a non-contact polarization-dependent optical coherence microscope (POCM) was developed to enable non-invasive in vivo imaging of human corneal microstructures. The system integrated quarter-wave plates into the sample and reference arms of the interferometer to enable deeper penetration of light in tissues as well as mitigate the strong specular reflection from the corneal surface. A common-path approach was adopted to enable control over the polarization in a free space configuration, thus alleviating the need for a broadband polarization-maintained fiber. The POCM achieved volumetric imaging of corneal microstructures, including endothelial cells over a field of view 0.5 × 0.5 mm2 with an almost isotropic resolution of ∼2.2 µm and a volume (500 × 500 × 2048 voxels) rate of 1 Hz. A self-interference approach between the corneal surface and underlying layers was also developed to lessen the corneal curvature and axial motion artifacts, thus enabling high-resolution imaging of microstructures in the anterior cornea, including squamous epithelial cells, wing epithelial cells, basal epithelial cells, sub-basal nerve plexus, and stromal keratocytes.

Optical Analysis and Reappraisal of the Peripheral Light Focusing Theory of Nasal Pterygia Formation.

Purpose: Pterygia are much more common nasally than temporally. Ultraviolet (UV) radiation is a major risk factor. Coroneo proposed that the nasal preference is caused by the "peripheral light focusing effect," (PLF), in which UV at an oblique angle passes through temporal cornea and is concentrated on and damages nasal limbal stem cells. This study evaluates whether the PLF is sufficient to explain the nasal preference. Methods: Whereas Coroneo and colleagues derived the maximum PLF intensity gain (UV concentration factor) as a function of incident angle (i.e., different nasal limbal positions were used for different incident angles) the current analysis derived intensity gain at a fixed position such at the nasal corneo-limbal junction (CLJ). This provided a measure of the total PLF irradiation at this position, which was compared to total direct irradiation of nasal and temporal limbus at the corresponding positions (e.g., CLJs). In Part 1, analysis was performed like that of Coroneo, using horizontally incident UV; in Part 2, the analysis was extended to include incident rays above and below the horizontal. Results: In both part 1 and part 2 of the study, the limbal UV irradiation of the nasal limbus from the PLF was not sufficient to explain the strong nasal location preference of pterygia. Conclusions: The analysis calls into question the PLF explanation of nasal location preference. Other explanations of the nasal preference, and of pterygium pathogenesis, should be considered, such as temporal to nasal tear flow carrying substances such as cytokines to the nasal limbus.

Quantitative assessment of human donor corneal endothelium with Gabor domain optical coherence microscopy.

We report on a pathway for Gabor domain optical coherence microscopy (GD-OCM)-based metrology to assess the donor's corneal endothelial layers ex vivo. Six corneas from the Lions Eye Bank at Albany and Rochester were imaged with GD-OCM. The raw 3-D images of the curved corneas were flattened using custom software to enhance the 2-D visualization of endothelial cells (ECs); then the ECs within a circle of 500-µm-diameter were analyzed using a custom corner method and a cell counting plugin in ImageJ. The EC number, EC area, endothelial cell density (ECD), and polymegethism (CV) were quantified in five different locations for each cornea. The robustness of the method (defined as the repeatability of measurement together with interoperator variability) was evaluated by independently repeating the entire ECD measurement procedure six times by three different examiners. The results from the six corneas show that the current modality reproduces the ECDs with a standard deviation of 2.3% of the mean ECD in every location, whereas the mean ECD across five locations varies by 5.1%. The resolution and imaging area provided through the use of GD-OCM may help to ultimately better assess the quality of donor corneas in transplantation.

Capabilities of Gabor-domain optical coherence microscopy for the assessment of corneal disease.

To identify the microstructural modification of the corneal layers during the course of the disease, optical technologies have been pushing the boundary of innovation to achieve cellular resolution of deep layers of the cornea. Gabor-domain optical coherence microscopy (GD-OCM), an optical coherence tomography-based technique that can achieve an isotropic of ∼2-µm resolution over a volume of 1 mm × 1 mm × 1.2 mm, was developed to investigate the microstructural modifications of corneal layers in four common corneal diseases. Since individual layer visualization without cutting through several layers is challenging due to corneal curvature, a flattening algorithm was developed to remove the global curvature of the endothelial layer and display the full view of the endothelium and Descemet's membrane in single en face images. As a result, GD-OCM revealed the qualitative changes in size and reflectivity of keratocytes in Fuchs endothelial corneal dystrophy (FECD), which varied by the degree of disease. More importantly, elongated shape and hyperactivation characteristics of keratocytes, associated with the early development of guttae, appeared to start in the posterior stroma very early in the disease process and move toward the anterior stroma during disease progression. This work opens a venue into the pathogenesis of FECD.

MEMS-based handheld scanning probe with pre-shaped input signals for distortion-free images in Gabor-domain optical coherence microscopy.

High-speed scanning in optical coherence tomography (OCT) often comes with either compromises in image quality, the requirement for post-processing of the acquired images, or both. We report on distortion-free OCT volumetric imaging with a dual-axis micro-electro-mechanical system (MEMS)-based handheld imaging probe. In the context of an imaging probe with optics located between the 2D MEMS and the sample, we report in this paper on how pre-shaped open-loop input signals with tailored non-linear parts were implemented in a custom control board and, unlike the sinusoidal signals typically used for MEMS, achieved real-time distortion-free imaging without post-processing. The MEMS mirror was integrated into a compact, lightweight handheld probe. The MEMS scanner achieved a 12-fold reduction in volume and 17-fold reduction in weight over a previous dual-mirror galvanometer-based scanner. Distortion-free imaging with no post-processing with a Gabor-domain optical coherence microscope (GD-OCM) with 2 µm axial and lateral resolutions over a field of view of 1 × 1 mm2 is demonstrated experimentally through volumetric images of a regular microscopic structure, an excised human cornea, and in vivo human skin.

Optical Assessment of Soft Contact Lens Edge-Thickness.

PURPOSE: To assess the edge shape of soft contact lenses using Gabor-Domain Optical Coherence Microscopy (GD-OCM) with a 2-µm imaging resolution in three dimensions and to generate edge-thickness profiles at different distances from the edge tip of soft contact lenses. METHODS: A high-speed custom-designed GD-OCM system was used to produce 3D images of the edge of an experimental soft contact lens (Bausch + Lomb, Rochester, NY) in four different configurations: in air, submerged into water, submerged into saline with contrast agent, and placed onto the cornea of a porcine eyeball. An algorithm to compute the edge-thickness was developed and applied to cross-sectional images. The proposed algorithm includes the accurate detection of the interfaces between the lens and the environment, and the correction of the refraction error. RESULTS: The sharply defined edge tip of a soft contact lens was visualized in 3D. Results showed precise thickness measurement of the contact lens edge profile. Fifty cross-sectional image frames for each configuration were used to test the robustness of the algorithm in evaluating the edge-thickness at any distance from the edge tip. The precision of the measurements was less than 0.2 µm. CONCLUSIONS: The results confirmed the ability of GD-OCM to provide high-definition images of soft contact lens edges. As a nondestructive, precise, and fast metrology tool for soft contact lens measurement, the integration of GD-OCM in the design and manufacturing of contact lenses will be beneficial for further improvement in edge design and quality control. In the clinical perspective, the in vivo evaluation of the lens fitted onto the cornea will advance our understanding of how the edge interacts with the ocular surface. The latter will provide insights into the impact of long-term use of contact lenses on the visual performance.

Real Time Gabor-Domain Optical Coherence Microscopy for 3D Imaging.

Fast, robust, nondestructive 3D imaging is needed for the characterization of microscopic tissue structures across various clinical applications. A custom microelectromechanical system (MEMS)-based 2D scanner was developed to achieve, together with a multi-level GPU architecture, 55 kHz fast-axis A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) custom instrument. GD-OCM yields high-definition micrometer-class volumetric images. A dynamic depth of focusing capability through a bio-inspired liquid lens-based microscope design, as in whales' eyes, was developed to enable the high definition instrument throughout a large field of view of 1 mm3 volume of imaging. Developing this technology is prime to enable integration within the workflow of clinical environments. Imaging at an invariant resolution of 2 µm has been achieved throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. Volumetric scans of human skin in vivo and an excised human cornea are presented.

Optimization of galvanometer scanning for optical coherence tomography.

We study experimentally the effective duty cycle of galvanometer-based scanners (GSs) with regard to three main parameters of the scanning process: theoretical/imposed duty cycle (of the input signal), scan frequency, and scan amplitude. Sawtooth and triangular input signals for the device are considered. The effects of the mechanical inertia of the oscillatory element of the GS are analyzed and their consequences are discussed in the context of optical coherence tomography (OCT) imaging. When the theoretical duty cycle and the scan amplitude are increased to the limit, the saturation of the device is demonstrated for a useful range of scan frequencies by direct measurement of the position of the galvomirror. Investigations of OCT imaging of large samples also validate this saturation, as examplified by the gaps/blurred portions obtained between neighboring images when using both triangular and sawtooth scanning at high scan frequencies. For this latter aspect, the necessary overlap between neighboring B-scans, and therefore between the corresponding volumetric reconstructions of the sample, are evaluated and implemented with regard to the same parameters of the scanning process. OCT images that are free of these artifacts are thus obtained.

Assessing microstructures of the cornea with Gabor-domain optical coherence microscopy: pathway for corneal physiology and diseases.

Gabor-domain optical coherence microscopy (GD-OCM) was applied ex vivo in the investigation of corneal cells and their surrounding microstructures with particular attention to the corneal endothelium. Experiments using fresh pig eyeballs, excised human corneal buttons from patients with Fuchs' endothelial dystrophy (FED), and healthy donor corneas were conducted. Results show in a large field of view (1 mm×1 mm) high definition images of the different cell types and their surrounding microstructures through the full corneal thickness at both the central and peripheral locations of porcine corneas. Particularly, an image of the endothelial cells lining the bottom of the cornea is highlighted. As compared to healthy human corneas, the corneas of individuals with FED show characteristic microstructural alterations of the Descemet's membrane and increased size and number of keratocytes. The GD-OCM-based imaging system developed may constitute a novel tool for corneal imaging and disease diagnosis. Also, importantly, it may provide insights into the mechanism of corneal physiology and pathology, particularly in diseases of the corneal endothelium.

Parallelized multi-graphics processing unit framework for high-speed Gabor-domain optical coherence microscopy.

Gabor-domain optical coherence microscopy (GD-OCM) is a volumetric high-resolution technique capable of acquiring three-dimensional (3-D) skin images with histological resolution. Real-time image processing is needed to enable GD-OCM imaging in a clinical setting. We present a parallelized and scalable multi-graphics processing unit (GPU) computing framework for real-time GD-OCM image processing. A parallelized control mechanism was developed to individually assign computation tasks to each of the GPUs. For each GPU, the optimal number of amplitude-scans (A-scans) to be processed in parallel was selected to maximize GPU memory usage and core throughput. We investigated five computing architectures for computational speed-up in processing 1000×1000 A-scans. The proposed parallelized multi-GPU computing framework enables processing at a computational speed faster than the GD-OCM image acquisition, thereby facilitating high-speed GD-OCM imaging in a clinical setting. Using two parallelized GPUs, the image processing of a 1×1×0.6 mm3 skin sample was performed in about 13 s, and the performance was benchmarked at 6.5 s with four GPUs. This work thus demonstrates that 3-D GD-OCM data may be displayed in real-time to the examiner using parallelized GPU processing.

Measurement of a multi-layered tear film phantom using optical coherence tomography and statistical decision theory.

To extend our understanding of tear film dynamics for the management of dry eye disease, we propose a method to optically sense the tear film and estimate simultaneously the thicknesses of the lipid and aqueous layers. The proposed method, SDT-OCT, combines ultra-high axial resolution optical coherence tomography (OCT) and a robust estimator based on statistical decision theory (SDT) to achieve thickness measurements at the nanometer scale. Unlike conventional Fourier-domain OCT where peak detection of layers occurs in Fourier space, in SDT-OCT thickness is estimated using statistical decision theory directly on the raw spectra acquired with the OCT system. In this paper, we demonstrate in simulation that a customized OCT system tailored to ~1 µm axial point spread function (FWHM) in the corneal tissue, combined with the maximum-likelihood estimator, can estimate thicknesses of the nanometer-scale lipid and micron-scale aqueous layers of the tear film, simultaneously, with nanometer precision. This capability was validated in experiments using a physical phantom that consists of two layers of optical coatings that mimic the lipid and aqueous layers of the tear film.

Analysis and adaptation of convolution algorithms to reconstruct extended objects in digital holography.

This paper discusses convolution algorithms to reconstruct off-axis digital holograms. The problem of convolution is addressed by considering the spatial spectral properties of digital holograms, especially the unusual localization property of the Fourier spectrum of the hologram, in regard to the physical object space. After deriving the sampling requirements for the transfer functions, three approaches are considered with the concept of spatial bandwidth extension: zero padding, spectrum scanning, and adjustable magnification. The theoretical discussion is completed by experimental illustrations that enable the algorithms to be objectively compared.

Experimental and theoretical investigation of the pixel saturation effect in digital holography.

This paper presents an experimental investigation and an analytical modeling of the nonlinear pixel saturation effect in digital off-axis holography. The theoretical analysis is based on a semiempirical modeling and supported by the experimental analysis. Taking into account the nonlinearity of the phenomenon, an exponential law for the high-order harmonic amplitude is proposed and validated by the experimental results. The conclusion of this analysis is that the saturation effect can be described by the use of a linear operator that involves autoconvolution of the initial object wave, even though the saturation phenomenon is nonlinear.

Digital holographic reconstruction of a local object field using an adjustable magnification.

In the research of digital holography, this paper presents a numerical method using an adjustable magnification for local object field reconstruction together with experiment verification. The method first designs a spherical wave according to the given magnification to illuminate the digital hologram, then through a Fourier transform of diffraction, it calculates the reconstructed image plane. Afterward, a filtering window is set in the image plane to extract the image of the local object field, and then the object field reached hologram plane is formed using diffraction's inverse operation. Finally, the object field is reconstructed through diffraction's angular spectrum theory.

Real-time three-sensitivity measurements based on three-color digital Fresnel holographic interferometry.

This Letter presents a method for real-time 3D measurements based on three-color digital holographic interferometry. The optical setup is considerably simplified, since the reference beams are combined into a unique beam. A convolution algorithm allows the three monochrome images to be superposed to provide simultaneous full-field 3D measurements. Experimental results confirm the suitability of the proposed method.

Method of digital holographic recording and reconstruction using a stacked color image sensor.

We discuss a method to record and reconstruct color holograms by using a stack of photodiode sensors associated to a one-way reference beam. The reconstruction algorithm follows a convolution strategy in which a transverse magnification leads to the full reconstruction of the object in the reconstructed horizon. The transverse magnification of the object depends on the curvature of the reference wave. Analysis of the spatial resolution indicates that it is linked to the transversal magnification but that no extra information is gained or lost in the process. Experimental results confirm the validity of the proposed approach for two-color digital holography. The error due to spectral mixing is investigated and found to be quite irrelevant compared to the range of the phase measurement.

Spatial bandwidth extended reconstruction for digital color Fresnel holograms.

This paper presents a reconstruction algorithm based on the convolution formula of diffraction which uses the Fresnel impulse response of free space propagation. The bandwidth of the reconstructing convolution kernel is extended to the one of the object in order to allow the direct reconstruction of objects with size quite larger than the recording area. The spatial bandwidth extension is made possible by the use of a numerical spherical wave as a virtual reconstructing wave, thus modifying the virtual reconstruction distance and increasing the kernel bandwidth. Experimental results confirm the suitability of the proposed method in the case of the simultaneous recording of two-color digital holograms by using a spatial color multiplexing scheme.

Digital holographic reconstruction of large objects using a convolution approach and adjustable magnification.

We present a numerical method for reconstructing large objects using a convolution method with an adjustable magnification. The method is based on the image locations and magnification relations of holography when the illuminating beam is a spherical wavefront. A modified version of the angular spectrum transfer function is proposed that allows the filtering in the spatial frequency spectrum. Experimental results confirm the suitability of the proposed method.