Multiple scattering is one of the main factors that limits the penetration depth of optical coherence tomography (OCT) in scattering samples. We propose a method termed multi-focus averaging (MFA) to suppress the multiple-scattering signals and improve the image contrast of OCT in deep regions. The MFA method captures multiple OCT volumes with various focal positions and averages them in complex form after correcting the varying defocus through computational refocusing. Because the multiple-scattering takes different trajectories among the different focal position configurations, this averaging suppresses the multiple-scattering signal. Meanwhile, the single-scattering takes a consistent trajectory regardless of the focal position configuration and is not suppressed. Hence, the MFA method improves the ratio between the single-scattering signal and multiple-scattering signal, resulting in an enhancement in the image contrast. A scattering phantom and a postmortem zebrafish were measured to validate the proposed method. The results showed that the contrast of intensity images of both the phantom and zebrafish were improved using the MFA method, such that they were better than the contrast provided by the standard single focus averaging method. The MFA method provides a cost-effective solution for contrast enhancement through multiple-scattering reduction in tissue imaging using OCT systems.
This study aims at demonstrating label-free drug-response-patterns assessment of different tumor spheroids and drug types by dynamic optical coherence tomography (D-OCT). The study involved human breast cancer (MCF-7) and colon cancer (HT-29) spheroids. The MCF-7 and HT-29 spheroids were treated with paclitaxel (Taxol; PTX) and the active metabolite of irinotecan SN-38, respectively. The drugs were applied with 0 (control), 0.1, 1, and 10 µM concentrations and the treatment durations were 1, 3, and 6 days. A swept-source OCT microscope equipped with a repeated raster scanning protocol was used to scan the spheroids. Logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDS[Formula: see text]) algorithms were used to visualize the tumor spheroid dynamics. LIV and OCDS[Formula: see text] images visualized different response patterns of the two types of spheroids. In addition, spheroid morphology, LIV, and OCDS[Formula: see text] quantification showed different time-courses among the spheroid and drug types. These results may indicate different action mechanisms of the drugs. The results showed the feasibility of D-OCT for the evaluation of drug response patterns of different cell spheroids and drug types and suggest that D-OCT can perform anti-cancer drug testing.
Breast Neoplasms , Colonic Neoplasms , Humans , Female , Tomography, Optical Coherence , Algorithms , Drug Evaluation , Irinotecan/pharmacology , Paclitaxel
A new formulation of the lateral imaging process of point-scanning optical coherence tomography (OCT) and a new differential contrast method designed by using this formulation are presented. The formulation is based on a mathematical sample model called the dispersed scatterer model (DSM), in which the sample is represented as a material with a spatially slowly varying refractive index and randomly distributed scatterers embedded in the material. It is shown that the formulation represents a meaningful OCT image and speckle as two independent mathematical quantities. The new differential contrast method is based on complex signal processing of OCT images, and the physical and numerical imaging processes of this method are jointly formulated using the same theoretical strategy as in the case of OCT. The formula shows that the method provides a spatially differential image of the sample structure. This differential imaging method is validated by measuring in vivo and in vitro samples.
An organoid is a three-dimensional (3D) in vitro cell culture emulating human organs. We applied 3D dynamic optical coherence tomography (DOCT) to visualize the intratissue and intracellular activities of human induced pluripotent stem cells (hiPSCs)-derived alveolar organoids in normal and fibrosis models. 3D DOCT data were acquired with an 840-nm spectral domain optical coherence tomography with axial and lateral resolutions of 3.8 µm (in tissue) and 4.9 µm, respectively. The DOCT images were obtained by the logarithmic-intensity-variance (LIV) algorithm, which is sensitive to the signal fluctuation magnitude. The LIV images revealed cystic structures surrounded by high-LIV borders and mesh-like structures with low LIV. The former may be alveoli with a highly dynamics epithelium, while the latter may be fibroblasts. The LIV images also demonstrated the abnormal repair of the alveolar epithelium.
Breast cancer is a leading cause of death in female patients worldwide. Further research is needed to get a deeper insight into the mechanisms involved in the development of this devastating disease and to find new therapy strategies. The zebrafish is an established animal model, especially in the field of oncology, which has shown to be a promising candidate for pre-clinical research and precision-based medicine. To investigate cancer growth in vivo in zebrafish, one approach is to explore xenograft tumor models. In this article, we present the investigation of a juvenile xenograft zebrafish model using a Jones matrix optical coherence tomography (JM-OCT) prototype. Immunosuppressed wild-type fish at 1-month post-fertilization were injected with human breast cancer cells and control animals with phosphate buffered saline in the tail musculature. In a longitudinal study, the scatter, polarization, and vasculature changes over time were investigated and quantified in control versus tumor injected animals. A significant decrease in birefringence and an increase in scattering signal was detected in tumor injected zebrafish in comparison to the control once. This work shows the potential of JM-OCT as a non-invasive, label-free, three-dimensional, high-resolution, and tissue-specific imaging tool in pre-clinical cancer research based on juvenile zebrafish models.
Breast Neoplasms , Tomography, Optical Coherence , Animals , Disease Models, Animal , Female , Heterografts , Humans , Longitudinal Studies , Tomography, Optical Coherence/methods , Zebrafish
Label-free metabolic imaging of non-alcoholic fatty liver disease (NAFLD) mouse liver is demonstrated ex vivo by dynamic optical coherence tomography (OCT). The NAFLD mouse is a methionine choline-deficient (MCD)-diet model, and two mice fed the MCD diet for 1 and 2 weeks are involved in addition to a normal-diet mouse. The dynamic OCT is based on repeating raster scan and logarithmic intensity variance (LIV) analysis that enables volumetric metabolic imaging with a standard-speed (50,000 A-lines/s) OCT system. Metabolic domains associated with lipid droplet accumulation and inflammation are clearly visualized three-dimensionally. Particularly, the normal-diet liver exhibits highly metabolic vessel-like structures of peri-vascular hepatic zones. The 1-week MCD-diet liver shows ring-shaped highly metabolic structures formed with lipid droplets. The 2-week MCD-diet liver exhibits fragmented vessel-like structures associated with inflammation. These results imply that volumetric LIV imaging is useful for visualizing and assessing NAFLD abnormalities.
[This corrects the article on p. 2975 in vol. 13.].
Here we demonstrate a long-depth-of-focus imaging method using polarization sensitive optical coherence tomography (PS-OCT). This method involves a combination of Fresnel-diffraction-model-based phase sensitive computational refocusing and Jones-matrix based PS-OCT (JM-OCT). JM-OCT measures four complex OCT images corresponding to four polarization channels. These OCT images are computationally refocused as preserving the mutual phase consistency. This method is validated using a static phantom, postmortem zebrafish, and ex vivo porcine muscle samples. All the samples demonstrated successful computationally-refocused birefringence and degree-of-polarization-uniformity (DOPU) images. We found that defocusing induces polarization artifacts, i.e., incorrectly high birefringence values and low DOPU values, which are substantially mitigated by computational refocusing.
The zebrafish is a valuable vertebrate animal model in pre-clinical cancer research. A Jones matrix optical coherence tomography (JM-OCT) prototype operating at 1310 nm and an intensity-based spectral-domain OCT setup at 840 nm were utilized to investigate adult wildtype and a tumor-developing zebrafish model. Various anatomical features were characterized based on their inherent scattering and polarization signature. A motorized translation stage in combination with the JM-OCT prototype enabled large field-of-view imaging to investigate adult zebrafish in a non-destructive way. The diseased animals exhibited tumor-related abnormalities in the brain and near the eye region. The scatter intensity, the attenuation coefficients and local polarization parameters such as the birefringence and the degree of polarization uniformity were analyzed to quantify differences in tumor versus control regions. The proof-of-concept study in a limited number of animals revealed a significant decrease in birefringence in tumors found in the brain and near the eye compared to control regions. The presented work showed the potential of OCT and JM-OCT as non-destructive, high-resolution, and real-time imaging modalities for pre-clinical research based on zebrafish.
SIGNIFICANCE: The scattering and polarization characteristics of various organs of in vivo wildtype zebrafish in three development stages were investigated using a non-destructive and label-free approach. The presented results showed a promising first step for the usability of Jones-matrix optical coherence tomography (JM-OCT) in zebrafish-based research. AIM: We aim to visualize and quantify the scatter and polarization signatures of various zebrafish organs for larvae, juvenile, and young adult animals in vivo in a non-invasive and label-free way. APPROACH: A custom-built polarization-sensitive JM-OCT setup in combination with a motorized translation stage was utilized to investigate live zebrafish. Depth-resolved scattering (intensity and attenuation coefficient) and polarization (birefringence and degree of polarization uniformity) properties were analyzed. OCT angiography (OCT-A) was utilized to investigate the vasculature label-free and non-destructively. RESULTS: The scatter and polarization signatures of the zebrafish organs such as the eye, gills, and muscles were investigated. The attenuation coefficient and birefringence changes between 1- and 2-month-old animals were evaluated in selected organs. OCT-A revealed the vasculature of in vivo larvae and juvenile zebrafish in a label-free manner. CONCLUSIONS: JM-OCT offers a rapid, label-free, non-invasive, tissue specific, and three-dimensional imaging tool to investigate in vivo processes in zebrafish in various development stages.
Tomography, Optical Coherence , Zebrafish , Animals , Birefringence , Refraction, Ocular
The zebrafish is an established vertebrae model in the field of biomedical research. With its small size, rapid maturation time and semi-transparency at early development stages, it has proven to be an important animal model, especially for high-throughput studies. Three-dimensional, high-resolution, non-destructive and label-free imaging techniques are perfectly suited to investigate these animals over various development stages. Optical coherence tomography (OCT) is an interferometric-based optical imaging technique that has revolutionized the diagnostic possibilities in the field of ophthalmology and has proven to be a powerful tool for many microscopic applications. Recently, OCT found its way into state-of-the-art zebrafish-based research. This review article gives an overview and a discussion of the relevant literature and an outlook for this emerging field.
Achieving high resolution in optical coherence tomography typically requires the continuous extension of the spectral bandwidth of the light source. This work demonstrates an alternative approach: combining two discrete spectral windows located in the visible spectrum with a trained conditional generative adversarial network (cGAN) to reconstruct a high-resolution image equivalent to that generated using a continuous spectral band. The cGAN was trained using OCT image pairs acquired with the continuous and discontinuous visible range spectra to learn the relation between low- and high-resolution data. The reconstruction performance was tested using 6000 B-scans of a layered phantom, micro-beads and ex-vivo mouse ear tissue. The resultant cGAN-generated images demonstrate an image quality and axial resolution which approaches that of the high-resolution system.
We present a completely label-free three-dimensional (3D) optical coherence tomography (OCT)-based tissue dynamics imaging method for visualization and quantification of the metabolic and necrotic activities of tumor spheroid. Our method is based on a custom 3D scanning protocol that is designed to capture volumetric tissue dynamics tomography images only in a few tens of seconds. The method was applied to the evaluation of a tumor spheroid. The time-course viability alteration and anti-cancer drug response of the spheroid were visualized qualitatively and analyzed quantitatively. The similarity between the OCT-based dynamics images and fluorescence microscope images was also demonstrated.
Cataracts are the leading cause of blindness worldwide. Here we propose optical coherence tomography (OCT) as a quantitative method for investigating cataracts. OCT provides volumetric and non-invasive access to the lens and makes it possible to rapidly observe the formation of opacifications in animal models such as mice. We compared the performance of two different wavelengths - 1060â nm and 1310â nm - for OCT imaging in cataract research. In addition, we present multi-contrast OCT capable of mapping depth-resolved scattering and average anterior cortical attenuation properties of the crystalline lens and quantitatively characterize induced cataract development in the mouse eye. Lastly, we also propose a novel method based on the retinal OCT projection image for quantifying and mapping opacifications in the lens, which showed a good correlation with scattering and attenuation characteristics simultaneously analyzed during the process of cataract formation in the lens.
Vascular leakage plays a key role in vision-threatening retinal diseases such as diabetic retinopathy and age-related macular degeneration. Fluorescence angiography is the current gold standard for identification of leaky vasculature in vivo, however it lacks depth resolution, providing only 2D images that complicate precise identification and localization of pathological vessels. Optical coherence tomography (OCT) has been widely adopted for clinical ophthalmology due to its high, micron-scale resolution and rapid volumetric scanning capabilities. Nevertheless, OCT cannot currently identify leaky blood vessels. To address this need, we have developed a new method called exogenous contrast-enhanced leakage OCT (ExCEL-OCT) which identifies the diffusion of tracer particles around leaky vasculature following injection of a contrast agent. We apply this method to a mouse model of retinal neovascularization and demonstrate high-resolution 3D vascular leakage measurements in vivo for the first time.
Polarization-sensitive optical coherence tomography (PS-OCT) enables three-dimensional imaging of biological tissues based on the inherent contrast provided by scattering and polarization properties. In fibrous tissue such as the white matter of the brain, PS-OCT allows quantitative mapping of tissue birefringence. For the popular PS-OCT layout using a single circular input state, birefringence measurements are based on a straight-forward evaluation of phase retardation data. However, the accuracy of these measurements strongly depends on the signal-to-noise ratio (SNR) and is prone to mapping artifacts when the SNR is low. Here we present a simple yet effective approach for improving the accuracy of PS-OCT phase retardation and birefringence measurements. By performing a noise bias correction of the detected OCT signal amplitudes, the impact of the noise floor on retardation measurements can be markedly reduced. We present simulation data to illustrate the influence of the noise bias correction on phase retardation measurements and support our analysis with real-world PS-OCT image data.
Artifacts , Tomography, Optical Coherence , Birefringence , Neuroimaging , Signal-To-Noise Ratio
Significance: Amyloid-beta ( A - ß ) plaques are pathological protein deposits formed in the brain of Alzheimer's disease (AD) patients upon disease progression. Further research is needed to elucidate the complex underlying mechanisms involved in their formation using label-free, tissue preserving, and volumetric techniques. Aim: The aim is to achieve a one-to-one correlation of optical coherence tomography (OCT) data to histological micrographs of brain tissue using 1060-nm swept source OCT. Approach: A - ß plaques were investigated in ex-vivo AD brain tissue using OCT with the capability of switching between two magnifications. For the exact correlation to histology, a 3D-printed tool was designed to generate samples with parallel flat surfaces. Large field-of-view (FoV) and sequentially high-resolution volumes at different locations were acquired. The large FoV served to align the OCT to histology images; the high-resolution images were used to visualize fine details. Results: The instrument and the presented method enabled an accurate correlation of histological micrographs with OCT data. A - ß plaques were identified as hyperscattering features in both FoV OCT modalities. The plaques identified in volumetric OCT data were in good agreement with immunohistochemically derived micrographs. Conclusion: OCT combined with the 3D-printed tool is a promising approach for label-free, nondestructive, volumetric, and fast tissue analysis.
Fluorescence-guided surgery is a state-of-the-art approach for intraoperative imaging during neurosurgical removal of tumor tissue. While the visualization of high-grade gliomas is reliable, lower grade glioma often lack visible fluorescence signals. Here, we present a hybrid prototype combining visible light optical coherence microscopy (OCM) and high-resolution fluorescence imaging for assessment of brain tumor samples acquired by 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. OCM provides high-resolution information of the inherent tissue scattering and absorption properties of tissue. We here explore quantitative attenuation coefficients derived from volumetric OCM intensity data and quantitative high-resolution 5-ALA fluorescence as potential biomarkers for tissue malignancy including otherwise difficult-to-assess low-grade glioma. We validate our findings against the gold standard histology and use attenuation and fluorescence intensity measures to differentiate between tumor core, infiltrative zone and adjacent brain tissue. Using large field-of-view scans acquired by a near-infrared swept-source optical coherence tomography setup, we provide initial assessments of tumor heterogeneity. Finally, we use cross-sectional OCM images to train a convolutional neural network that discriminates tumor from non-tumor tissue with an accuracy of 97%. Collectively, the present hybrid approach offers potential to translate into an in vivo imaging setup for substantially improved intraoperative guidance of brain tumor surgeries.
Diagnostic classification techniques used to diagnose cataracts, the world's leading cause of blindness, are currently based on subjective methods. Here, we present optical coherence tomography as a noninvasive tool for volumetric visualization of lesions formed in the crystalline lens. A custom-made swept-source optical coherence tomography (SS-OCT) system was utilized to investigate the murine crystalline lens. In addition to imaging cataractous lesions in aged wildtype mice, we studied the structure and shape of cataracts in a mouse model of Alzheimer's disease. Hyperscattering opacifications in the crystalline lens were observed in both groups. Post mortem histological analysis were performed to correlate findings in the anterior and posterior part of the lens to 3D OCT in vivo imaging. Our results showcase the capability of OCT to rapidly visualize cataractous lesions in the murine lens and suggest that OCT might be a valuable tool that provides additional insight for preclinical studies of cataract formation.
Significance. Recent Alzheimer's disease (AD) patient studies have focused on retinal analysis, as the retina is the only part of the central nervous system that can be imaged noninvasively by optical methods. However, as this is a relatively new approach, the occurrence and role of retinal pathological features are still debated. Aim. The retina of an APP/PS1 mouse model was investigated using multicontrast optical coherence tomography (OCT) in order to provide a documentation of what was observed in both transgenic and wild-type mice. Approach. Both eyes of 24 APP/PS1 transgenic mice (age: 45 to 104 weeks) and 15 age-matched wild-type littermates were imaged by the custom-built OCT system. At the end of the experiment, retinas and brains were harvested from a subset of the mice (14 transgenic, 7 age-matched control) in order to compare the in vivo results to histological analysis and to quantify the cortical amyloid beta plaque load. Results. The system provided a combination of standard reflectivity data, polarization-sensitive data, and OCT angiograms. Qualitative and quantitative information from the resultant OCT images was extracted on retinal layer thickness and structure, presence of hyper-reflective foci, phase retardation abnormalities, and retinal vasculature. Conclusions. Although multicontrast OCT revealed abnormal structural properties and phase retardation signals in the retina of this APP/PS1 mouse model, the observations were very similar in transgenic and control mice.
