Hyperreflective Dots in Central Fovea Visualized by a Novel Application of Visible-Light Optical Coherence Tomography.

Visible-light optical coherence tomography (vis-OCT) is a novel noninvasive retinal imaging system that offers improved resolution compared to conventional near-infrared (NIR) OCT systems. Here, we utilized vis-OCT to produce fibergrams (vis-OCTF) for the first time in human patients, enabling en face visualization and precise quantification of hyperreflective dots in the central fovea in two patients. We also directly compare the imaging qualities of conventional vis-OCT and NIR-OCT. Vis-OCT generated a 3 × 3 mm2 en face image with an impressive axial resolution of 1.3 µm, whereas NIR-OCT produced an en face image with a larger field of view (FOV) (9 × 9 mm2) but a lower resolution of 7.0 µm. Moreover, vis-OCTF unveiled clear images of hyperreflective dots in the fovea of both patients, which were not discernible in the NIR-OCT en face images. Foveal dots have often been linked to several age-related and pathological conditions. The high-resolution images generated by vis-OCTF enable more precise characterization of changes in retinal sublayers within the central fovea.

Freeform robotic optical coherence tomography beyond the optical field-of-view limit.

Imaging complex, non-planar anatomies with optical coherence tomography (OCT) is limited by the optical field of view (FOV) in a single volumetric acquisition. Combining linear mechanical translation with OCT extends the FOV but suffers from inflexibility in imaging non-planar anatomies. We report the freeform robotic OCT to fill this gap. To address challenges in volumetric reconstruction associated with the robotic movement accuracy being two orders of magnitudes worse than OCT imaging resolution, we developed a volumetric registration algorithm based on simultaneous localization and mapping (SLAM) to overcome this limitation. We imaged the entire aqueous humor outflow pathway, whose imaging has the potential to customize glaucoma surgeries but is typically constrained by the FOV, circumferentially in mice as a test. We acquired volumetric OCT data at different robotic poses and reconstructed the entire anterior segment of the eye. The reconstructed volumes showed heterogeneous Schlemm's canal (SC) morphology in the reconstructed anterior segment and revealed a segmental nature in the circumferential distribution of collector channels (CC) with spatial features as small as a few micrometers.

Adaptive balanced detection spectral domain optical coherence tomography.

Balanced detection optical coherence tomography (BD-OCT) enables near-shot noise-limited imaging by suppressing wavelength-dependent relative intensity noise (RIN) originating from the light source. In spectral-domain BD-OCT (SD-BD-OCT), the level of RIN suppression relies on the co-registration accuracy of the spectra simultaneously captured by two independent spectrometers. However, existing matching methods require careful pre-calibration using a RIN-dominated dataset or subjective post-processing using a signal-dominated dataset. We developed an adaptive subpixel matching approach, referred to as adaptive balance, that can be applied to any SD-BD-OCT dataset regardless of RIN or signal level without the need for pre-calibration. We showed that adaptive balance performed comparable to or better than reported methods by imaging phantoms with varying spectrometer camera gain, exposure time, and supercontinuum laser repetition rate. We further demonstrated the benefits of adaptive balance in human retinal imaging.

Adaptive spectroscopic visible-light optical coherence tomography for clinical retinal oximetry.

BACKGROUND: Retinal oxygen saturation (sO2) provides essential information about the eye's response to pathological changes that can result in vision loss. Visible-light optical coherence tomography (vis-OCT) is a noninvasive tool that has the potential to measure retinal sO2 in a clinical setting. However, its reliability is currently limited by unwanted signals referred to as spectral contaminants (SCs), and a comprehensive strategy to isolate true oxygen-dependent signals from SCs in vis-OCT is lacking. METHODS: We develop an adaptive spectroscopic vis-OCT (ADS-vis-OCT) technique that can adaptively remove SCs and accurately measure sO2 under the unique conditions of each vessel. We also validate the accuracy of ADS-vis-OCT using ex vivo blood phantoms and assess its repeatability in the retina of healthy volunteers. RESULTS: In ex vivo blood phantoms, ADS-vis-OCT agrees with a blood gas machine with only a 1% bias in samples with sO2 ranging from 0% to 100%. In the human retina, the root mean squared error between sO2 values in major arteries measured by ADS-vis-OCT and a pulse oximeter is 2.1% across 18 research participants. Additionally, the standard deviations of repeated ADS-vis-OCT measurements of sO2 values in smaller arteries and veins are 2.5% and 2.3%, respectively. Non-adaptive methods do not achieve comparable repeatabilities from healthy volunteers. CONCLUSIONS: ADS-vis-OCT effectively removes SCs from human images, yielding accurate and repeatable sO2 measurements in retinal arteries and veins with varying diameters. This work could have important implications for the clinical use of vis-OCT to manage eye diseases.

Numerous diseases that cause blindness are associated with disrupted oxygen consumption in the retina, the part of the eye that senses light. This highlights the importance of accurately measuring oxygen consumption in the clinic. To address this challenge, we developed a method to analyze images of the retina which have been collected using visible-light optical coherence tomography, a non-invasive imaging method. Our approach achieves accurate oxygen level measurements in blood samples and in healthy volunteers. With further testing, our approach may prove useful in the clinical management of several diseases that cause blindness, allowing clinicians to more accurately diagnose disease and monitor the health of the eye.

In Vivo Sublayer Analysis of Human Retinal Inner Plexiform Layer Obtained by Visible-Light Optical Coherence Tomography.

Purpose: Growing evidence suggests that dendrite retraction or degeneration in a subpopulation of the retinal ganglion cells (RGCs) may precede detectable soma abnormalities and RGC death in glaucoma. Visualization of the lamellar structure of the inner plexiform layer (IPL) could advance clinical management and fundamental understanding of glaucoma. We investigated whether visible-light optical coherence tomography (vis-OCT) could detect the difference in the IPL sublayer thicknesses between small cohorts of healthy and glaucomatous subjects. Method: We imaged nine healthy and five glaucomatous subjects with vis-OCT. Four of the healthy subjects were scanned three times each in two separate visits, and five healthy and five glaucoma subjects were scanned three times during a single visit. IPL sublayers were manually segmented using averaged A-line profiles. Results: The mean ages of glaucoma and healthy subjects are 59.6 ± 13.4 and 45.4 ± 14.4 years (P = 0.02.) The visual field mean deviations (MDs) are -26.4 to -7.7 dB in glaucoma patients and -1.6 to 1.1 dB in healthy subjects (P = 0.002). Median coefficients of variation (CVs) of intrasession repeatability for the entire IPL and three sublayers are 3.1%, 5.6%, 6.9%, and 5.6% in healthy subjects and 1.8%, 6.0%, 7.7%, and 6.2% in glaucoma patients, respectively. The mean IPL thicknesses are 36.2 ± 1.5 µm in glaucomatous and 40.1 ± 1.7 µm in healthy eyes (P = 0.003). Conclusions: IPL sublayer analysis revealed that the middle sublayer could be responsible for the majority of IPL thinning in glaucoma. Vis-OCT quantified IPL sublayers with good repeatability in both glaucoma and healthy subjects.

High-Speed Balanced-Detection Visible-Light Optical Coherence Tomography in the Human Retina Using Subpixel Spectrometer Calibration.

Increases in speed and sensitivity enabled rapid clinical adoption of optical coherence tomography (OCT) in ophthalmology. Recently, visible-light OCT (vis-OCT) achieved ultrahigh axial resolution, improved tissue contrast, and provided new functional imaging capabilities, demonstrating the potential to improve clinical care further. However, limited speed and sensitivity caused by the high relative intensity noise (RIN) in supercontinuum lasers impeded the clinical adoption of vis-OCT. To overcome these limitations, we developed balanced-detection vis-OCT (BD-vis-OCT), which uses two calibrated spectrometers to cancel RIN and other noises. We analyzed the RIN to achieve robust subpixel calibration between the two spectrometers and showed that BD-vis-OCT reduced the A-line noise floor by up to 20.5 dB. Metrics comparing signal-to-noise-ratios showed similar image qualities across multiple reference arm powers, a hallmark of operation near the shot-noise limit. We imaged healthy human retinas at an A-line rate of 125 kHz and a field-of-view up to 10 mm ×4 mm. We found that BD-vis-OCT revealed retinal anatomical features previously obscured by the noise floor.

Intrinsic spectrally-dependent background in spectroscopic visible-light optical coherence tomography.

Visible-light optical coherence tomography (vis-OCT) has enabled new spectroscopic applications, such as retinal oximetry, as a result of increased optical absorption and scattering contacts in biological tissue and improved axial resolution. Besides extracting tissue properties from back-scattered light, spectroscopic analyses must consider spectral alterations induced by image reconstruction itself. We investigated an intrinsic spectral bias in the background noise floor, which is hereby referred to as the spectrally-dependent background (SDBG). We developed an analytical model to predict the SDBG-induced bias and validated this model using numerically simulated and experimentally acquired data. We found that SDBG systemically altered the measured spectra of blood in human retinal vessels in vis-OCT, as compared to literature data. We provided solutions to quantify and compensate for SDBG in retinal oximetry. This work is particularly significant for clinical applications of vis-OCT.

Visible-Light Optical Coherence Tomography Fibergraphy for Quantitative Imaging of Retinal Ganglion Cell Axon Bundles.

Purpose: To develop a practical technique for visualizing and quantifying retinal ganglion cell (RGC) axon bundles in vivo. Methods: We applied visible-light optical coherence tomography (vis-OCT) to image the RGC axon bundles, referred to as vis-OCT fibergraphy, of healthy wild-type C57BL/6 mice. After vis-OCT imaging, retinas were flat-mounted, immunostained with anti-beta-III tubulin (Tuj1) antibody for RGC axons, and imaged with confocal microscopy. We quantitatively compared the RGC axon bundle networks imaged by in vivo vis-OCT and ex vivo confocal microscopy using semi-log Sholl analysis. Results: Side-by-side comparison of ex vivo confocal microscopy and in vivo vis-OCT confirmed that vis-OCT fibergraphy captures true RGC axon bundle networks. The semi-log Sholl regression coefficients extracted from vis-OCT fibergrams (3.7 ± 0.8 mm-1) and confocal microscopy (3.6 ± 0.3 mm-1) images also showed good agreement with each other (n = 6). Conclusions: We demonstrated the feasibility of using vis-OCT fibergraphy to visualize RGC axon bundles. Further applying Sholl analysis has the potential to identify biomarkers for non-invasively assessing RGC health. Translational Relevance: Our novel technique for visualizing and quantifying RGC axon bundles in vivo provides a potential measurement tool for diagnosing and tracking the progression of optic neuropathies.

Spectrally dependent roll-off in visible-light optical coherence tomography.

Recent development of visible-light optical coherence tomography (vis-OCT) has introduced new applications for noninvasive spectroscopic imaging. However, the measured spectra may be altered by spectrally dependent roll-off (SDR). We formulated a mathematical model for SDR that accounted for nonuniform wavenumber spacing, optical aberrations, and misalignments in the spectrometer. We simulated SDR based on this model and found strong agreement with measurements from a vis-OCT system. We verified that SDR altered spectroscopic measurements of fully oxygenated blood. We corrected these alterations by normalizing each spectrally dependent A-line by the measured SDR of the spectrometer. Our investigations of SDR are critical for informing OCT spectrometer design, alignment, and spectroscopic measurements.

Longitudinal deep-brain imaging in mouse using visible-light optical coherence tomography through chronic microprism cranial window.

We longitudinally imaged both the superficial and deep cortical microvascular networks in brains of healthy mice and in a mouse model of stroke in vivo using visible-light optical coherence tomography (vis-OCT). We surgically implanted a microprism in mouse brains sealed by a chronic cranial window. The microprism enabled vis-OCT to image the entire depth of the mouse cortex. Following microprism implantation, we imaged the mice for 28 days and found that that it took around 15 days for both the superficial and deep cortical microvessels to recover from the implantation surgery. After the brains recovered, we introduced ischemic strokes by transient middle cerebral artery occlusion (tMCAO). We monitored the strokes for up to 60 days and observed different microvascular responses to tMCAO at different cortical depths in both the acute and chronic phases of the stroke. This work demonstrates that the combined microprism and cranial window is well-suited for longitudinal investigation of cortical microvascular disorders using vis-OCT.

Speckle reduction in visible-light optical coherence tomography using scan modulation.

We present a technique to reduce speckle in visible-light optical coherence tomography (vis-OCT) that preserves fine structural details and is robust against sample motion. Specifically, we locally modulate B-scans orthogonally to their axis of acquisition. Such modulation enables acquisition of uncorrelated speckle patterns from similar anatomical locations, which can be averaged to reduce speckle. To verify the effectiveness of speckle reduction, we performed in-vivo retinal imaging using modulated raster and circular scans in both mice and humans. We compared speckle-reduced vis-OCT images with the images acquired with unmodulated B-scans from the same anatomical locations. We compared contrast-to-noise ratio (CNR) and equivalent number of looks (ENL) to quantify the image quality enhancement. Speckle-reduced images showed up to a 2.35-dB improvement in CNR and up to a 3.1-fold improvement in ENL with more discernable anatomical features using eight modulated A-line averages at a 25-kHz A-line rate.

Designing visible-light optical coherence tomography towards clinics.

BACKGROUND: The capabilities of visible-light optical coherence tomography (vis-OCT) in noninvasive anatomical and functional retinal imaging have been demonstrated by multiple groups in both rodents and healthy human subjects. Translating laboratory prototypes to an integrated clinical-environment-friendly system is required to explore the full potential of vis-OCT in disease management. METHODS: We developed and optimized a portable vis-OCT system for human retinal imaging in clinical settings. We acquired raster- and circular-scan images from both healthy and diseased human eyes. RESULTS: The new vis-OCT provided high-quality retinal images of both subjects without any known eye diseases and patients with various retinal diseases, including retinal occlusive disease and diabetic retinopathy (DR) over a broad range of ages. CONCLUSIONS: A newly designed vis-OCT system is sufficiently optimized to be suited for routine patients' examinations in clinics. Vis-OCT has the potential to add new anatomical and functional imaging capabilities to ophthalmic clinical care.

Dual-wavelength photothermal optical coherence tomography for imaging microvasculature blood oxygen saturation.

A swept-source dual-wavelength photothermal (DWP) optical coherence tomography (OCT) system is demonstrated for quantitative imaging of microvasculature oxygen saturation. DWP-OCT is capable of recording three-dimensional images of tissue and depth-resolved phase variation in response to photothermal excitation. A 1,064-nm OCT probe and 770-nm and 800-nm photothermal excitation beams are combined in a single-mode optical fiber to measure microvasculature hemoglobin oxygen saturation (SO(2)) levels in phantom blood vessels with a range of blood flow speeds (0 to 17 mm/s). A 50-µm-diameter blood vessel phantom is imaged, and SO(2) levels are measured using DWP-OCT and compared with values provided by a commercial oximeter at various blood oxygen concentrations. The influences of blood flow speed and mechanisms of SNR phase degradation on the accuracy of SO(2) measurement are identified and investigated.

Use of near-infrared luminescent gold nanoclusters for detection of macrophages.

We determined the effect of aggregation and coating thickness of gold on the luminescence of nanoparticles engulfed by macrophages and in gelatin phantoms. Thin gold-coated iron oxide nanoclusters (nanoroses) have been developed to target macrophages to provide contrast enhancement for near-infrared optical imaging applications. We compare the brightness of nanoroses luminescent emissions in response to 635 nm laser excitation to other nanoparticles including nanoshells, nanorods, and Cy5 conjugated iron oxide nanoparticles. Luminescent properties of all these nanoparticles were investigated in monomeric and aggregated form in gelatin phantoms and primary macrophage cell cultures using confocal microscopy. Aggregation of the gold nanoparticles increased luminescence emission and correlated with increased surface mass of gold per nanoparticle (nanoshells 37 ± 14.30 × 10(-3) brightness with 1.23 × 10(-4) wt of gold (g)/nanoparticle versus original nanorose 1.45 ± 0.37 × 10(-3) with 2.10 × 10(-16) wt of gold/nanoparticle, p<0.05). Nanoshells showed greater luminescent intensity than original nanoroses or Cy5 conjugated iron oxide nanoparticles when compared as nanoparticles per macrophage (38 ± 10 versus 11 ± 2.8 versus 17 ± 6.5, p<0.05, respectively, ANOVA), but showed relatively poor macrophage uptake (1025 ± 128 versus 7549 ± 236 versus 96,000 nanoparticles/cell, p<0.05, student t-test nanoshells versus nanoroses). Enhancement of gold fluorescent emissions by nanoparticles can be achieved by reducing the thickness of the gold coating, by clustering the gold on the surface of the nanoparticles (nanoshells), and by clustering the gold nanoparticles themselves.

In vivo depth-resolved oxygen saturation by Dual-Wavelength Photothermal (DWP) OCT.

Microvasculature hemoglobin oxygen saturation (SaO2) is important in the progression of various pathologies. Non-invasive depth-resolved measurement of SaO2 levels in tissue microvasculature has the potential to provide early biomarkers and a better understanding of the pathophysiological processes allowing improved diagnostics and prediction of disease progression. We report proof-of-concept in vivo depth-resolved measurement of SaO(2) levels in selected 30 µm diameter arterioles in the murine brain using Dual-Wavelength Photothermal (DWP) Optical Coherence Tomography (OCT) with 800 nm and 770 nm photothermal excitation wavelengths. Depth location of back-reflected light from a target arteriole was confirmed using Doppler and speckle contrast OCT images. SaO(2) measured in a murine arteriole with DWP-OCT is linearly correlated (R(2)=0.98) with systemic SaO(2) values recorded by a pulse-oximeter. DWP-OCT are steadily lower (10.1%) than systemic SaO(2) values except during pure oxygen breathing. DWP-OCT is insensitive to OCT intensity variations and is a candidate approach for in vivo depth-resolved quantitative imaging of microvascular SaO(2) levels.

Lamina-specific anatomic magnetic resonance imaging of the human retina.

PURPOSE: Magnetic resonance imaging (MRI) of the human retina faces two major challenges: eye movement and hardware limitation that could preclude human retinal MRI with adequate spatiotemporal resolution. This study investigated eye-fixation stability and high-resolution anatomic MRI of the human retina on a 3-Tesla (T) MRI scanner. Comparison was made with optical coherence tomography (OCT) on the same subjects. METHODS: Eye-fixation stability of protocols used in MRI was evaluated on four normal volunteers using an eye tracker. High-resolution MRI (100 × 200 × 2000 µm) protocol was developed on a 3-T scanner. Subjects were instructed to maintain stable eye fixation on a target with cued blinks every 8 seconds during MRI. OCT imaging of the retina was performed. Retinal layer thicknesses measured with MRI and OCT were analyzed for matching regions of the same eyes close to the optic nerve head. RESULTS: The temporal SDs of the horizontal and vertical displacements were 78 ± 51 and 130 ± 51 µm (±SD, n = 4), respectively. MRI detected three layers within the human retina, consistent with MRI findings in rodent, feline, and baboon retinas. The hyperintense layer 1 closest to the vitreous likely consisted of nerve fiber, ganglion cell, and inner nuclear layer; the hypointense layer 2, the outer nuclear layer and the inner and outer segments; and the hyperintense layer 3, the choroid. The MRI retina/choroid thickness was 711 ± 37 µm, 19% (P < 0.05) thicker than OCT thickness (579 ± 34 µm). CONCLUSIONS: This study reports high-resolution MRI of lamina-specific structures in the human retina. These initial results are encouraging. Further improvement in spatiotemporal resolution is warranted.

Depth-resolved blood oxygen saturation measurement by dual-wavelength photothermal (DWP) optical coherence tomography.

Non-invasive depth-resolved measurement of hemoglobin oxygen saturation (SaO(2)) levels in discrete blood vessels may have implications for diagnosis and treatment of various pathologies. We introduce a novel Dual-Wavelength Photothermal (DWP) Optical Coherence Tomography (OCT) for non-invasive depth-resolved measurement of SaO(2) levels in a blood vessel phantom. DWP OCT SaO(2) is linearly correlated with blood-gas SaO(2) measurements. We demonstrate 6.3% precision in SaO(2) levels measured a phantom blood vessel using DWP-OCT with 800 and 765 nm excitation wavelengths. Sources of uncertainty in SaO(2) levels measured with DWP-OCT are identified and characterized.

Comparison of pulsed photothermal radiometry, optical coherence tomography and ultrasound for melanoma thickness measurement in PDMS tissue phantoms.

Melanoma accounts for 75% of all skin cancer deaths. Pulsed photothermal radiometry (PPTR), optical coherence tomography (OCT) and ultrasound (US) are non-invasive imaging techniques that may be used to measure melanoma thickness, thus, determining surgical margins. We constructed a series of PDMS tissue phantoms simulating melanomas of different thicknesses. PPTR, OCT and US measurements were recorded from PDMS tissue phantoms and results were compared in terms of axial imaging range, axial resolution and imaging time. A Monte Carlo simulation and three-dimensional heat transfer model was constructed to simulate PPTR measurement. Experimental results show that PPTR and US can provide a wide axial imaging range (75 µm-1.7 mm and 120-910 µm respectively) but poor axial resolution (75 and 120 µm respectively) in PDMS tissue phantoms, while OCT has the most superficial axial imaging range (14-450 µm) but highest axial resolution (14 µm). The Monte Carlo simulation and three-dimensional heat transfer model give good agreement with PPTR measurement. PPTR and US are suited to measure thicker melanoma lesions (>400 µm), while OCT is better to measure thin melanoma lesions (<400 µm).

Depth resolved photothermal OCT detection of macrophages in tissue using nanorose.

Application of photothermal Optical Coherence Tomography (OCT) to detect macrophages in ex vivo rabbit arteries which have engulfed nanoclusters of gold coated iron oxide (nanorose) is reported. Nanorose engulfed by macrophages associated with atherosclerotic lesions in rabbit arteries absorb incident laser (800nm) energy and cause optical pathlength (OP) variation which is measured using photothermal OCT. OP variation in polydimethyl siloxane tissue phantoms containing varying concentrations of nanorose match values predicted from nanoparticle and material properties. Measurement of OP variation in rabbit arteries in response to laser excitation provides an estimate of nanorose concentration in atherosclerotic lesions of 2.5x10(9) particles/ml. OP variation in atherosclerotic lesions containing macrophages taking up nanorose has a different magnitude and profile from that observed in control thoracic aorta without macrophages and is consistent with macrophage presence as identified with RAM-11 histology staining. Our results suggest that tissue regions with macrophages taking up nanorose can be detected using photothermal OCT.

Effect on blood glucose monitoring of skin pressure exerted by an optical coherence tomography probe.

We proposed to use optical coherence tomography (OCT) for continuous noninvasive blood glucose monitoring, and recently we significantly improved the sensitivity of this technique. The accuracy of OCT glucose monitoring is limited by several factors, including variation of tissue pressure exerted by the OCT probe. We studied the influence of high (>10 kPa) and low (<1 kPa) pressure levels on OCT blood glucose monitoring. We showed that controlling external pressure to <1 kPa substantially improved the accuracy and reproducibility of OCT-based glucose monitoring.