Adaptive optics optical coherence tomography (AO-OCT) has allowed for the reliable 3-D imaging of individual retinal cells. The current AO-OCT systems are limited to tabletop implementation due to their size and complexity. This work describes the design and implementation of the first dual modality handheld AO-OCT (HAOOCT) and scanning laser ophthalmoscope (SLO) probe to extend AO-OCT imaging to previously excluded patients. Simultaneous SLO imaging allows for tracking of imaging features for HAOOCT localization. Pilot experiments on stabilized and recumbent adults using HAOOCT, weighing only 665 grams, revealed the 3-D photoreceptor structure for the first time using a handheld AO-OCT/SLO device.
The incorporation of adaptive optics (AO) technology into ophthalmic imaging systems has enhanced the understanding of retinal structure and function and the progression of various retinal diseases in adults by allowing for the dynamic correction of ocular and/or system aberrations. However, the in vivo visualization of important human retinal microanatomy, including cone photoreceptors, has been largely limited to fully cooperative subjects who are able to fixate and/or sit upright for extended imaging sessions in large tabletop AO systems. Previously, we developed the first handheld AO scanning laser ophthalmoscope capable of 2-D imaging of cone photoreceptors in supine adults and infants. In this work, we present the design and fabrication of the first handheld AO optical coherence tomography (HAOOCT) probe capable of collecting high-resolution volumetric images of the human retina. We designed custom optomechanics to build a spectral domain OCT system with a compact form factor of 22 cm × 18 cm × 5.2 cm and a total weight of 630 grams. The OCT imaging channel has a theoretical lateral resolution of 2.26 μm over a 1.0° × 1.0° field of view and an axial resolution of 4.01 μm. Stabilized imaging of healthy human adult volunteers revealed the 3-D photoreceptor structure and retinal pigment epithelium cells. HAOOCT was then deployed in handheld operation to image photoreceptors in upright and recumbent adults, indicating its potential to extend AO-OCT to previously excluded patient populations.
Adaptive optics scanning laser ophthalmoscopy (AOSLO) has advanced the study of retinal structure and function by enabling in vivo imaging of individual photoreceptors. Most implementations of AOSLOs are large, complex tabletop systems, thereby preventing high quality photoreceptor imaging of patients who are unable to sit upright and/or fixate for an imaging session. We have previously addressed this limitation in the clinical translation of AOSLO by developing the first confocal handheld AOSLO (HAOSLO) capable of cone photoreceptor visualization in adults and infants. However, confocal AOSLO images suffer from imaging artifacts and the inability to detect remnant cone structure, leading to ambiguous or potentially misleading results. Recently, it has been shown that non-confocal split-detection (SD) AOSLO images, created by the collection of multiply backscattered light, enables more reliable studies of retinal photoreceptors by providing images of the cone inner segment. In this paper, we detail the extension of our HAOSLO probe to enable multi-channel light collection resulting in the first ever multimodal handheld AOSLO (M-HAOSLO). Imaging sessions were conducted on two dilated, healthy human adult volunteers, and M-HAOSLO images taken in handheld operation mode reveal the cone photoreceptor mosaic. Aside from being the first miniaturized and portable implementation of a SD AOSLO system, M-HAOSLO relies on sensorless optimization of the wavefront to correct aberrations. Thus, we also show the first ever SD images collected after correction of the eye’s estimated wavefront.
Conventional scanning laser ophthalmoscopy (SLO) utilizes a finite collection pinhole at a retinal conjugate plane to strongly reject out-of-focus light while primarily transmitting the in-focus, retinal backscattered signal. However, to improve lateral resolution, a sub-Airy disk collection pinhole is necessary, which drastically reduces the signal-to-noise ratio (SNR) of the system and is thus not commonly employed. Recently, an all-optical, super-resolution microscopy technique known as optical photon reassignment (OPRA) microscopy (also known as re-scan confocal microscopy) has been developed to bypass this fundamental tradeoff between resolution and SNR in confocal microscopy. We present a methodology and system design for obtaining super resolution in retinal imaging by combining the concepts of SLO and OPRA microscopy. The resolution improvement of the system was quantified using a 1951 USAF target at a telecentric intermediate image plane. Retinal images from human volunteers were acquired with this system both with and without using the OPRA technique to demonstrate the resolution improvement when imaging parafoveal cone photoreceptors. Finally, we quantified the resolution improvement in the retina by analyzing the radially averaged power spectrum of the retinal images.
The human retina is composed of several layers, visible by in vivo optical coherence tomography (OCT) imaging. To enhance diagnostics of retinal diseases, several algorithms have been developed to automatically segment one or more of the boundaries of these layers. OCT images are corrupted by noise, which is frequently the result of the detector noise and speckle, a type of coherent noise resulting from the presence of several scatterers in each voxel. However, it is unknown what the empirical distribution of noise in each layer of the retina is, and how the magnitude and distribution of the noise affects the lower bounds of segmentation accuracy. Five healthy volunteers were imaged using a spectral domain OCT probe from Bioptigen, Inc, centered at 850nm with 4.6µm full width at half maximum axial resolution. Each volume was segmented by expert manual graders into nine layers. The histograms of intensities in each layer were then fit to seven possible noise distributions from the literature on speckle and image processing. Using these empirical noise distributions and empirical estimates of the intensity of each layer, the Cramer-Rao lower bound (CRLB), a measure of the variance of an estimator, was calculated for each boundary layer. Additionally, the optimum bias of a segmentation algorithm was calculated, and a corresponding biased CRLB was calculated, which represents the improved performance an algorithm can achieve by using prior knowledge, such as the smoothness and continuity of layer boundaries. Our general mathematical model can be easily adapted for virtually any OCT modality.
In vivo photoreceptor imaging has enhanced the way vision scientists and ophthalmologists understand the retinal structure, function, and etiology of numerous retinal pathologies. However, the complexity and large footprint of current systems capable of resolving photoreceptors has limited imaging to patients who are able to sit in an upright position and fixate for several minutes. Unfortunately, this excludes an important fraction of patients including bedridden patients, small children, and infants. Here, we show that our dual-modality, high-resolution handheld probe with a weight of only 94 g is capable of visualizing photoreceptors in supine children. Our device utilizes a microelectromechanical systems (MEMS) scanner and a novel telescope design to achieve over an order of magnitude reduction in size compared to similar systems. The probe has a 7° field of view and a lateral resolution of 8 µm. The optical coherence tomography (OCT) system has an axial resolution of 7 µm and a sensitivity of 101 dB. High definition scanning laser ophthalmoscopy (SLO) and OCT images were acquired from children ranging from 14 months to 12 years of age with and without pathology during examination under anesthesia in the operating room. Parafoveal cone imaging was shown using the SLO arm of this device without adaptive optics using a 3° FOV for the first time in children under 4 years old. This work lays the foundation for pediatric research, which will improve understanding of retinal development, maldevelopment and early onset of diseases at the cellular level during the beginning stages of human growth.
Handheld scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT) systems facilitate imaging of young children and subjects that have difficulty fixating. More compact and lightweight probes allow for better portability and increased comfort for the operator of the handheld probe. We describe a very compact, novel SLO and OCT handheld probe design. A single 2D microelectromechanical systems (MEMS) scanner and a custom optical design using a converging beam prior to the scanner permitted significant reduction in the system size. Our design utilized a combination of commercial and custom optics that were optimized in Zemax to achieve near diffraction-limited resolution of 8 μm over a 7° field of view. The handheld probe has a form factor of 7 x 6 x 2.5 cm and a weight of only 94 g, which is over an order of magnitude lighter than prior SLO-OCT handheld probes. Images were acquired from a normal subject with an incident power on the eye under the ANSI limit. With this device, which is the world’s lightest and smallest SLO-OCT system, we were able to visualize parafoveal cone photoreceptors and nerve fiber bundles without the use of adaptive optics.
Excessive nonspecific binding often occurs when labeling cells with immuno-labeled gold nanoparticles (IgG-AuNPs).
We have investigated the physical properties of IgG-AuNPs assembled with three different protocols in an attempt to
understand and eliminate this non-specific binding. One of these protocols involves conjugating the secondary antibody
AP124F via van der Waals (vdW) and/or electrostatic forces to the AuNPs, and the other two employ a PEG-linker,
OPSS-PEG-NHS (OPN). In all three protocols we follow with PEG-SH to provide protection against aggregation in
saline solution. OPN and PEG-SH chains of varying molecular weights were examined in different combinations to
determine the optimally protective layer. The hydrodynamic radius and surface plasmon resonance (SPR) were
monitored at each stage of assembly using a dynamic light scattering (DLS) instrument and spectrophotometer,
respectively. SPR measurements indicate a different physical structure near the gold surface when the PEG-linker is
bound to gold first and then bound to the antibody second (AP124F-[OPN-Au]) rather than vice versa ([AP124F-OPN]-
Au). These observed structural differences may lead to differences in the amount of non-specific binding observed when
immuno-labeling cells. SPR measurements also yielded a half-life of 27 minutes for the binding of the PEG-linker to the
surface of the AuNPs and a half-life of 133 minutes for the hydrolysis of the NHS functional groups on the OPN
molecule. These different reaction rates led us to add AP124F 40 minutes after the linker began binding to the AuNPs,
so that the antibody can bind covalently to the correct end of the OPN linker.
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