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.

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.

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.

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.

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.

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.

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.

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.

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.

Influence of osmolytes on in vivo glucose monitoring using optical coherence tomography.

Diabetes mellitus and its complications are the third leading cause of death in the world, exceeded only by cardiovascular disease and cancer. Tighter monitoring and control of blood glucose could minimize complications associated with diabetes. Recently, optical coherence tomography (OCT) for noninvasive glucose monitoring was proposed and tested in vivo. The aim of this work was to investigate the influence of changes in blood glucose concentration ([glu]) and sodium concentration ([Na+]) on the OCT signal. We also investigated the influence of other important analytes on the sensitivity of glucose monitoring with OCT. The experiments were carried out in anesthetized female pigs. The OCT images were acquired continuously from skin, while [glu] and [Na+] were experimentally varied within their physiological ranges. Correlations of the OCT signal slope with [glu] and [Na+] were studied at different tissue depths. The tissue area probed with OCT was marked and cut for histological examination. The correlation of blood [glu] and [Na+] with the OCT signal slope was observed in separate tissue layers. On average, equimolar changes in [glu] produced 2.26 +/- 1.15 greater alterations of the OCT signal slope than changes in [Na+]. Variation of concentrations of other analytes did not influence the OCT signal slope. The influence of [Na+] on relative changes in the OCT signal slope was generally less than [glu]-induced changes. OCT is a promising method for noninvasive glucose monitoring because of its ability to track the influence of changing [glu] on individual tissue layers.

In vivo study of glucose-induced changes in skin properties assessed with optical coherence tomography.

Recently, our in vivo studies demonstrated a strong correlation between blood glucose concentration and the slope of the optical coherence tomography (OCT) signal when the probing beam was scanned over a straight line. To improve the sensitivity of OCT for blood glucose monitoring, two-dimensional (2D) lateral scanning of the OCT probing beam was proposed. Depth-dependent changes in pig skin properties with variation of blood glucose concentration were revealed due to significant suppression of speckle noise and motion artefacts in 2D scanning mode. The correlation coefficient of the OCT signal slope with blood glucose concentration varied periodically in the range from -0.9 to +0.9 depending on depth. The period of variation of the correlation coefficient was 100-150 microm that corresponded to the distance between neighbour collagen bundles. We also observed a decrease of skin thickness by 10 +/- 7.5 microm with an increase of blood glucose concentration by 277 +/- 56 mg dl(-1). Mechanisms of glucose-induced changes in skin properties owing to tissue layer shift caused by dehydration associated with the glucose osmotic effect were considered.

Complementary use of cross-polarization and standard OCT for differential diagnosis of pathological tissues.

An experimental standard optical coherence tomography (OCT) setup that can be easily modified for cross-polarization OCT (CP OCT) operation has been developed to perform differential diagnosis of pathological tissues. The complementary use of CP OCT, a technique that provides a map of cross-polarization backscattering properties of an object being studied by means of low-coherence interferometry, and standard OCT imaging improves the specificity of diagnostics of pathological changes occurring in tissues. It is shown that healthy, neoplastic and scar tissues of the esophagus have different cross-polarization backscattering properties. A comparative analysis of CP OCT, OCT and histological images from one and the same tissue area has been made. A close correlation between the location of collagen fibers in biological tissue and signal intensity in CP OCT images is found.

Optical coherence tomography for in situ monitoring of laser corneal ablation.

OBJECTIVE: To improve the precision of refractive surgery, a new approach for determination of the removed corneal thickness profile in situ with laser ablation by optical coherence tomography (OCT) is developed. STUDY DESIGN/MATERIALS AND METHODS: The traditional method for precision (less than 10 microm) measurements of intraocular distances is based on the use of the reflected component of probing radiation. This component is characterized by a small range of operating angles between a probing beam and a normal to the surface under study. To enhance this range of operating angles we suggest using a light component backscattered from a biological object. This will enable precision measurements over the entire surface of the cornea without any changes in the orientation between a probing beam and the eye, a necessary condition for in situ monitoring of laser refraction correction in the eye. We suggest a specially developed algorithm of OCT signal processing to measure the corneal thickness by the backscattered light component for a single longitudinal scan (A scan). The corneal thickness profile is obtained by a series of such A scans acquired by successively scanning a probing beam along the corneal surface. The thickness profile of removed layer is determined by changes in the corneal thickness profile in the process of ablation. When the cornea is ablated by a beam with a fixed transverse profile, we propose using integral characteristics of the ablated layer profile, for example, the maximum ablation depth, as criteria of changes in refractive power of the eye. The measurement precision by these characteristics is considerably higher than by a single A scan. Since the cornea is a poorly scattering medium, the Fourier filtering is employed to increase reliability and precision of the method. Model experiments on monitoring the ablation process in a lavsan film and ex vivo human cornea are described. Preliminary experiments on in vivo measurements of human corneal thickness are performed. RESULTS: In model experiments the precision of measurement of laser ablation depth by one A scan was 5-20 microm, depending on the signal-to-noise ratio (SNR), whereas the precision of measurement of laser ablation depth as the integral characteristic of the ablated layer profile was 0.3-5 microm. The experimental results showed that at small SNR Fourier filtering might considerably increase reliability and precision of measurements. When SNR is high, the measurement precision does not change. The precision of measurements of the corneal thickness in preliminary in vivo experiments was higher than in ex vivo experiments. This factor is very promising for application of the method suggested herein in refractive surgery.

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.

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.