Deep learning-based label-free imaging of lymphatics and aqueous veins in the eye using optical coherence tomography.

We demonstrate an adaptation of deep learning for label-free imaging of the micro-scale lymphatic vessels and aqueous veins in the eye using optical coherence tomography (OCT). The proposed deep learning-based OCT lymphangiography (DL-OCTL) method was trained, validated and tested, using OCT scans (23 volumetric scans comprising 19,736 B-scans) from 11 fresh ex vivo porcine eyes with the corresponding vessel labels generated by a conventional OCT lymphangiography (OCTL) method based on thresholding with attenuation compensation. Compared to conventional OCTL, the DL-OCTL method demonstrates comparable results for imaging lymphatics and aqueous veins in the eye, with an Intersection over Union value of 0.79 ± 0.071 (mean ± standard deviation). In addition, DL-OCTL mitigates the imaging artifacts in conventional OCTL where the OCT signal modelling was corrupted by the tissue heterogeneity, provides ~ 10 times faster processing based on a rough comparison and does not require OCT-related knowledge for correct implementation as in conventional OCTL. With these favorable features, DL-OCTL promises to improve the practicality of OCTL for label-free imaging of lymphatics and aqueous veins for preclinical and clinical imaging applications.

Quantification of smooth muscle in human airways by polarization-sensitive optical coherence tomography requires correction for perichondrium.

Quantifying airway smooth muscle (ASM) in patients with asthma raises the possibility of improved and personalized disease management. Endobronchial polarization-sensitive optical coherence tomography (PS-OCT) is a promising quantitative imaging approach that is in the early stages of clinical translation. To date, only animal tissues have been used to assess the accuracy of PS-OCT to quantify absolute (rather than relative) ASM in cross sections with directly matched histological cross sections as validation. We report the use of whole fresh human and pig airways to perform a detailed side-by-side qualitative and quantitative validation of PS-OCT against gold-standard histology. We matched and quantified 120 sections from five human and seven pig (small and large) airways and linked PS-OCT signatures of ASM to the tissue structural appearance in histology. Notably, we found that human cartilage perichondrium can share with ASM the properties of birefringence and circumferential alignment of fibers, making it a significant confounder for ASM detection. Measurements not corrected for perichondrium overestimated ASM content several-fold (P < 0.001, paired t test). After careful exclusion of perichondrium, we found a strong positive correlation (r = 0.96, P < 0.00001) of ASM area measured by PS-OCT and histology, supporting the method's application in human subjects. Matching human histology further indicated that PS-OCT allows conclusions on the intralayer composition and in turn potential contractile capacity of ASM bands. Together these results form a reliable basis for future clinical studies.NEW & NOTEWORTHY Polarization-sensitive optical coherence tomography (PS-OCT) may facilitate in vivo measurement of airway smooth muscle (ASM). We present a quantitative validation correlating absolute ASM area from PS-OCT to directly matched histological cross sections using human tissue. A major confounder for ASM quantification was observed and resolved: fibrous perichondrium surrounding hyaline cartilage in human airways presents a PS-OCT signature similar to ASM for birefringence and optic axis orientation. Findings impact the development of automated methods for ASM segmentation.

In vivo burn scar assessment with speckle decorrelation and joint spectral and time domain optical coherence tomography.

Significance: Post-burn scars and scar contractures present significant challenges in burn injury management, necessitating accurate evaluation of the wound healing process to prevent or minimize complications. Non-invasive and accurate assessment of burn scar vascularity can offer valuable insights for evaluations of wound healing. Optical coherence tomography (OCT) and OCT angiography (OCTA) are promising imaging techniques that may enhance patient-centered care and satisfaction by providing detailed analyses of the healing process. Aim: Our study investigates the capabilities of OCT and OCTA for acquiring information on blood vessels in burn scars and evaluates the feasibility of utilizing this information to assess burn scars. Approach: Healthy skin and neighboring scar data from nine burn patients were obtained using OCT and processed with speckle decorrelation, Doppler OCT, and an enhanced technique based on joint spectral and time domain OCT. These methods facilitated the assessment of vascular structure and blood flow velocity in both healthy skin and scar tissues. Analyzing these parameters allowed for objective comparisons between normal skin and burn scars. Results: Our study found that blood vessel distribution in burn scars significantly differs from that in healthy skin. Burn scars exhibit increased vascularization, featuring less uniformity and lacking the intricate branching network found in healthy tissue. Specifically, the density of the vessels in burn scars is 67% higher than in healthy tissue, while axial flow velocity in burn scar vessels is 25% faster than in healthy tissue. Conclusions: Our research demonstrates the feasibility of OCT and OCTA as burn scar assessment tools. By implementing these technologies, we can distinguish between scar and healthy tissue based on its vascular structure, providing evidence of their practicality in evaluating burn scar severity and progression.

Ptychographic lens-less birefringence microscopy using a mask-modulated polarization image sensor.

Birefringence, an inherent characteristic of optically anisotropic materials, is widely utilized in various imaging applications ranging from material characterizations to clinical diagnosis. Polarized light microscopy enables high-resolution, high-contrast imaging of optically anisotropic specimens, but it is associated with mechanical rotations of polarizer/analyzer and relatively complex optical designs. Here, we present a form of lens-less polarization-sensitive microscopy capable of complex and birefringence imaging of transparent objects without an optical lens and any moving parts. Our method exploits an optical mask-modulated polarization image sensor and single-input-state LED illumination design to obtain complex and birefringence images of the object via ptychographic phase retrieval. Using a camera with a pixel size of 3.45 µm, the method achieves birefringence imaging with a half-pitch resolution of 2.46 µm over a 59.74 mm2 field-of-view, which corresponds to a space-bandwidth product of 9.9 megapixels. We demonstrate the high-resolution, large-area, phase and birefringence imaging capability of our method by presenting the phase and birefringence images of various anisotropic objects, including a monosodium urate crystal, and excised mouse eye and heart tissues.

Feasibility of deep learning-based polarization-sensitive optical coherence tomography angiography for imaging cutaneous microvasculature.

Polarization-sensitive optical coherence tomography (PS-OCT) measures the polarization states of the backscattered light from tissue that can improve angiography based on conventional optical coherence tomography (OCT). We present a feasibility study on PS-OCT integrated with deep learning for PS-OCT angiography (PS-OCTA) imaging of human cutaneous microvasculature. Two neural networks were assessed for PS-OCTA, including the residual dense network (RDN), which previously showed superior performance for angiography with conventional OCT and the upgraded grouped RDN (GRDN). We also investigated different protocols to process the multiple signal channels provided by the Jones matrices from the PS-OCT system to achieve optimal PS-OCTA performance. The training and testing of the deep learning-based PS-OCTA were performed using PS-OCT scans collected from 18 skin locations comprising 16,600 B-scan pairs. The results demonstrated a moderately improved performance of GRDN over RDN, and of the use of the combined signal from the Jones matrix elements over the separate use of the elements, as well as a similar image quality to that provided by speckle decorrelation angiography. GRDN-based PS-OCTA also showed ∼2-3 times faster processing and improved mitigation of tissue motion as compared to speckle decorrelation angiography, and enabled fully automatic processing. Deep learning-based PS-OCTA can be used for imaging cutaneous microvasculature, which may enable easy adoption of PS-OCTA for preclinical and clinical applications.

Non-Invasive Retinal Blood Vessel Wall Measurements with Polarization-Sensitive Optical Coherence Tomography for Diabetes Assessment: A Quantitative Study.

Diabetes affects the structure of the blood vessel walls. Since the blood vessel walls are made of birefringent organized tissue, any change or damage to this organization can be evaluated using polarization-sensitive optical coherence tomography (PS-OCT). In this paper, we used PS-OCT along with the blood vessel wall birefringence index (BBI = thickness/birefringence2) to non-invasively assess the structural integrity of the human retinal blood vessel walls in patients with diabetes and compared the results to those of healthy subjects. PS-OCT measurements revealed that blood vessel walls of diabetic patients exhibit a much higher birefringence while having the same wall thickness and therefore lower BBI values. Applying BBI to diagnose diabetes demonstrated high accuracy (93%), sensitivity (93%) and specificity (93%). PS-OCT measurements can quantify small changes in the polarization properties of retinal vessel walls associated with diabetes, which provides researchers with a new imaging tool to determine the effects of exercise, medication, and alternative diets on the development of diabetes.

Two concepts for ultra-high-resolution polarization-sensitive optical coherence tomography with a single camera.

Two designs with a multiplexed superluminescent diode for ultra-high-resolution spectral-domain polarization-sensitive optical coherence tomography (UHR-PS-OCT) are introduced. In the first design, a Wollaston prism separates orthogonal polarization states next to each other on one linescan camera; the other design uses a beam displacer to separate orthogonal states onto two lines of a linescan camera with multiple rows of detectors. The coherence lengths measured with the two systems were 3.6 µm and 2.9 µm (n=1.38), respectively. Measurements were collected from the fovea of a healthy subject, a healthy subject with astigmatism, and a patient with central serous retinopathy (CSR). A single volumetric scan provides double pass retardance induced by the retinal nerve fiber layer birefringence (RNFL) and its birefringence, the cumulative double pass retardance induced by the Henle fiber layer, and the retardance that is induced by the retinal pigment epithelium-Bruch's membrane complex. The high axial resolution in UHR-PS-OCT is particularly helpful for the measurements of thin retinal tissue, such as the RNFL in the fovea, where birefringence values of around 1°/µm were found. Tilting of the retina due to a CSR or by off centering the imaging beam in the pupil causes an artificial increase in the double pass retardance induced by the RNFL and Henle fiber layer.

Erratum: In vivo PS-OCT needle probe scan of human skeletal muscle: publisher's note.

[This corrects the article on p. 1386 in vol. 13, PMID: 35414965.].

In vivo PS-OCT needle probe scan of human skeletal muscle.

Polarization-sensitive optical coherence tomography (PS-OCT) derived birefringence values effectively identify skeletal muscle structural disruption due to muscular dystrophy and exercise-related muscle damage in animal models in ex vivo tissue. The purpose of this investigation was to determine if a PS-OCT needle probe inserted into the leg of a human subject could accurately identify various anatomical structures with implications for use as a diagnostic tool for the determination of skeletal muscle pathology. A healthy middle-aged subject participated in this study. A custom-built PS-OCT system was interfaced with a side-viewing fiber-optic needle probe inserted into the subject's vastus lateralis muscle via a motorized stage for 3D data acquisition via rotation and stepwise pullback. The deepest recorded PS-OCT images correspond to a depth of 6 mm beneath the dermis with structural images showing uniform, striated muscle tissue. Multiple highly birefringent band-like structures with definite orientation representing connective tissue of the superficial aponeurosis appeared as the depth of the needle decreased. Superficial to these structures the dominating appearance was that of adipose tissue and low birefringent but homogeneous scattering tissue. The data indicate that a PS-OCT needle probe can be inserted into live human skeletal muscle for the identification of relevant anatomical structures that could be utilized to diagnose significant skeletal muscle pathology.

Requirements and limitations of imaging airway smooth muscle throughout the lung in vivo.

Clinical visualization and quantification of the amount and distribution of airway smooth muscle (ASM) in the lungs of individuals with asthma has major implications for our understanding of airway wall remodeling as well as treatments targeted at the ASM. This paper theoretically investigates the feasibility of quantifying airway wall thickness (focusing on the ASM) throughout the lung in vivo by means of bronchoscopic polarization-sensitive optical coherence tomography (PS-OCT). Using extensive human biobank data from subjects with and without asthma in conjunction with a mathematical model of airway compliance, we define constraints that airways of various sizes pose to any endoscopic imaging technique and how this is impacted by physiologically relevant processes such as constriction, inflation and deflation. We identify critical PS-OCT system parameters and pinpoint parts of the airway tree that are conducive to successful quantification of ASM. We further quantify the impact of breathing and ASM contraction on the measurement error and recommend strategies for standardization and normalization.

Optical dosimeter for selective retinal therapy based on multi-port fiber-optic interferometry.

Selective retinal therapy (SRT) employs a micro-second short-pulse lasers to induce localized destruction of the targeted retinal structures with a pulse duration and power aimed at minimal damage to other healthy retinal cells. SRT has demonstrated a great promise in the treatment of retinal diseases, but pulse energy thresholds for effective SRT procedures should be determined precisely and in real time, as the thresholds could vary with disease status and patients. In this study, we present the use of a multi-port fiber-based interferometer (MFI) for highly sensitive real-time SRT monitoring. We exploit distinct phase differences among the fiber ports in the MFI to quantitatively measure localized fluctuations of complex-valued information during the SRT procedure. We evaluate several metrics that can be computed from the full complex-valued information and demonstrate that the complex contour integration is highly sensitive and most correlative to pulse energies, acoustic outputs, and cell deaths. The validity of our method was demonstrated on excised porcine retinas, with a sensitivity and specificity of 0.92 and 0.88, respectively, as compared with the results from a cell viability assay.

Polarization properties of retinal blood vessel walls measured with polarization sensitive optical coherence tomography.

A new method based on polarization-sensitive optical coherence tomography (PS-OCT) is introduced to determine the polarization properties of human retinal vessel walls, in vivo. Measurements were obtained near the optic nerve head of three healthy human subjects. The double pass phase retardation per unit depth (DPPR/UD), which is proportional to the birefringence, is higher in artery walls, presumably because of the presence of muscle tissue. Measurements in surrounding retinal nerve fiber layer tissue yielded lower DPPR/UD values, suggesting that the retinal vessel wall tissue near the optic nerve is not covered by retinal nerve fiber layer tissue (0.43°/µm vs. 0.77°/µm, respectively). Measurements were obtained from multiple artery-vein pairs, to quantify the different polarization properties. Measurements were taken along a section of the vessel wall, with changes in DPPR/UD up to 15%, while the vessel wall thickness remained relatively constant. A stationary scan pattern was applied to determine the influence of involuntary eye motion on the measurement, which was significant. Measurements were also analyzed by two examiners, with high inter-observer agreement. The measurement repeatability was determined with measurements that were acquired during multiple visits. An improvement in accuracy can be achieved with an ultra-broad-bandwidth PS-OCT system since it will provide more data points in-depth, which reduces the influence of discretization and helps to facilitate better fitting of the birefringence data.

Single-pixel, single-input-state polarization-sensitive wavefront imaging.

In this Letter, we describe a single-pixel polarization-sensitive imaging technique, capable of generating the birefringence map of a thin specimen by using single-pixel detectors. Spatially modulated light is circularly polarized to illuminate the specimen. The transmitted light through the specimen is then focused via a lens and measured by position-sensitive detectors in two orthogonal polarization channels. The measurement of the irradiance and centroid position of the optical focus and subsequent computations enable the production of polarization-dependent wavefront maps, which can then be utilized to reconstruct sample birefringence information. We demonstrate the feasibility of our method by measuring distribution of optic-axis orientation and phase retardation of various birefringent samples.

Vectorial birefringence imaging by optical coherence microscopy for assessing fibrillar microstructures in the cornea and limbus.

The organization of fibrillar tissue on the micrometer scale carries direct implications for health and disease but remains difficult to assess in vivo. Polarization-sensitive optical coherence tomography measures birefringence, which relates to the microscopic arrangement of fibrillar tissue components. Here, we demonstrate a critical improvement in leveraging this contrast mechanism by employing the improved spatial resolution of focus-extended optical coherence microscopy (1.4 µm axially in air and 1.6 µm laterally, over more than 70 µm depth of field). Vectorial birefringence imaging of sheep cornea ex vivo reveals its lamellar organization into thin sections with distinct local optic axis orientations, paving the way to resolving similar features in vivo.

Retinal layer thicknesses retrieved with different segmentation algorithms from optical coherence tomography scans acquired under different signal-to-noise ratio conditions.

Glaucomatous damage can be quantified by measuring the thickness of different retinal layers. However, poor image quality may hamper the accuracy of the layer thickness measurement. We determined the effect of poor image quality (low signal-to-noise ratio) on the different layer thicknesses and compared different segmentation algorithms regarding their robustness against this degrading effect. For this purpose, we performed OCT measurements in the macular area of healthy subjects and degraded the image quality by employing neutral density filters. We also analysed OCT scans from glaucoma patients with different disease severity. The algorithms used were: The Canon HS-100's built-in algorithm, DOCTRAP, IOWA, and FWHM, an approach we developed. We showed that the four algorithms used were all susceptible to noise at a varying degree, depending on the retinal layer assessed, and the results between different algorithms were not interchangeable. The algorithms also differed in their ability to differentiate between young healthy eyes and older glaucoma eyes and failed to accurately separate different glaucoma stages from each other.

Short-time series optical coherence tomography angiography and its application to cutaneous microvasculature.

We present a new optical coherence tomography (OCT) angiography method for imaging tissue microvasculature in vivo based on the characteristic frequency-domain flow signature in a short time series of a single voxel. The angiography signal is generated by Fourier transforming the OCT signal time series from a given voxel in multiple acquisitions and computing the average magnitude of non-zero (high-pass) frequency components. Larger temporal variations of the OCT signal caused by blood flow result in higher values of the average magnitude in the frequency domain compared to those from static tissue. Weighting of the signal by the inverse of the zero-frequency component (i.e., the sum of the OCT signal time series) improves vessel contrast in flow regions of low OCT signal. The method is demonstrated on a fabricated flow phantom and on human skin in vivo and, at only 5 time points per voxel, shows enhanced vessel contrast in comparison to conventional correlation mapping/speckle decorrelation and speckle variance methods.

Retinal imaging with optical coherence tomography and low-loss adaptive optics using a 2.8-mm beam size.

As data acquisition for retinal imaging with optical coherence tomography (OCT) becomes faster, efficient collection of photons becomes more important to maintain image quality. One approach is to use a larger aperture at the eye's pupil to collect more photons that have been reflected from the retina. A 2.8-mm beam diameter system with only seven reflecting surfaces was developed for low-loss retinal imaging. The larger beam size requires defocus and astigmatism correction, which was done in a closed loop adaptive optics method using a Shack-Hartmann wavefront sensor and a deformable mirror (DM) with 140 actuators and a ±2.75 µm stroke. This DM facilitates defocus correction ranging from approximately -3 D to +3 D. Comparing the new system with a standard 1.2-mm system on a model eye, a signal-to-noise gain of 4.5 dB and a 2.3 times smaller speckle size were measured. Measurements on the retinas of five subjects showed even better results, with increases in dynamic range up to 13 dB. Note that the new sample arm only occupies 30 cm × 60 cm, which makes it highly suitable for imaging in a clinical environment. Figure: B-scan images obtained over a width of 8 deg from the right eye of a 31-year-old Caucasian male. While the left side was imaged with a standard 1.2-mm OCT system, the right side was imaged with the 2.8-mm system. Both images were collected with the same integration time and incident power, after correction of aberrations. Using the dynamic range within the images, which is determined by comparing the highest pixel value to the noise floor, a difference in dynamic range of 10.8 dB was measured between the two systems.

Measuring polarization changes in the human outer retina with polarization-sensitive optical coherence tomography.

Morphological changes in the outer retina such as drusen are established biomarkers to diagnose age-related macular degeneration. However, earlier diagnosis might be possible by taking advantage of more subtle changes that accompany tissues that bear polarization-altering properties. To test this hypothesis, we developed a method based on polarization-sensitive optical coherence tomography with which volumetric data sets of the macula were obtained from 10 young (<25 years) and 10 older (>54 years) subjects. All young subjects and 5 of the older subjects had retardance values induced by the retinal pigment epithelium and Bruch's membrane (RPE-BM) complex that were just above the noise floor measurement (5°-13° at 840 nm). In contrast, elevated retardance, up to 180°, was observed in the other 5 older subjects. Analysis of the degree of polarization uniformity (DOPU) demonstrates that reduced DOPU (<0.4) in the RPE is associated with elevated double pass phase retardation (DPPR) below the RPE-BM complex, suggesting that the observed elevated DPPR in older subjects is the result of increased scattering or polarization scrambling. Collectively, our measurements show that the outer retina can undergo dramatic change in its polarization properties with age, and in some cases still retain its clinically normal appearance.

Optical coherence tomography with a 2.8-mm beam diameter and sensorless defocus and astigmatism correction.

An optical coherence tomography (OCT) system with a 2.8-mm beam diameter is presented. Sensorless defocus correction can be performed with a Badal optometer and astigmatism correction with a liquid crystal device. OCT B-scans were used in an image-based optimization algorithm for aberration correction. Defocus can be corrected from ? 4.3 ?? D to + 4.3 ?? D and vertical and oblique astigmatism from ? 2.5 ?? D to + 2.5 ?? D . A contrast gain of 6.9 times was measured after aberration correction. In comparison with a 1.3-mm beam diameter OCT system, this concept achieved a 3.7-dB gain in dynamic range on a model retina. Both systems were used to image the retina of a human subject. As the correction of the liquid crystal device can take more than 60 s, the subject's spectacle prescription was adopted instead. This resulted in a 2.5 times smaller speckle size compared with the standard OCT system. The liquid crystal device for astigmatism correction does not need a high-voltage amplifier and can be operated at 5 V. The correction device is small ( 9 ?? mm × 30 ?? mm × 38 ?? mm ) and can easily be implemented in existing designs for OCT.

Mueller-matrix modeling and characterization of a dual-crystal electro-optic modulator.

A general mathematical model based on Mueller-matrix calculation is presented to describe the optical behavior of a dual-crystal electro-optic modulator. The two crystals inside the modulator are oriented at ± 45° with respect to the horizontal, thereby cancelling natural birefringence and temperature-induced birefringence. We describe the behavior of the modulator as a function of the ellipticity of the crystals, the rotation angles of the crystals and the applied voltage. By fitting the measured data with a Mueller-matrix model that uses values for the ellipticity and orientation angles of the crystals, the simulated data and the experimental measurements could be matched. This Mueller-matrix includes physical properties of the thermally compensated electro optic modulator, and the matrix can be used in simulations where these device-specific properties are important, for instance in the modeling of a polarization-sensitive optical coherence tomography system.