Penta-Modal Imaging Platform with OCT- Guided Dynamic Focusing for Simultaneous Multimodal Imaging

Complex diseases, such as Alzheimer’s disease, are associated with sequences of changes in multiple disease-specific biomarkers. These biomarkers may show dynamic changes at specific stages of disease progression. Thus, testing/monitoring each biomarker may provide insight into specific disease-related processes, which can result in early diagnosis or even development of preventive measures. Obtaining a comprehensive information of biological tissues requires imaging of multiple optical contrasts, which is not typically offered by a single imaging modality. Thus, combining different contrast mechanisms to achieve simultaneous multimodal imaging is desirable. However, this process is highly challenging due to specific optical and hardware requirements for each optical imaging system. The objective of this dissertation is to develop a novel Penta-modal optical imaging system integrating photoacoustic microscopy (PAM), optical coherence tomography (OCT), optical Doppler tomography (ODT), OCT angiography (OCTA) and confocal fluorescence microscopy (CFM) in one platform providing comprehensive structural, functional, and molecular information of living biological tissues. The system can simultaneously image different biomarkers with a large field-of-view (FOV) and high-speed imaging. The large FOV and the high imaging speed is achieved by combining optical and mechanical scanning mechanisms. To compensate for an uneven surface of biological samples, which result in images with non-uniform resolution and low signal to noise ratio (SNR), we further develop a novel OCT-guided surface contour scanning methodology, a technique for adjusting objective lens focus to follow the contour of the sample surface, to provide a uniform spatial resolution and SNR across the region of interest (ROI). The imaging system was tested by imaging phantoms, ex vivo biological samples, and in vivo. The OCT-guided surface contour scanning methodology was utilized for imaging a leaf of purple queen plant, which resulted in a significant contrast improvement of 41% and 38% across a large imaging area for CFM and PAM, respectively. The nuclei and cells walls were also clearly observed in both images. In an in vivo imaging of the Swiss Webster mouse ear, our multimodal imaging system was able to provide images with uniform resolution in an FOV of 10 mm x 10 mm with an imaging time of around 5 minutes. In addition to measuring the blood flow in the mouse ear, the system also successfully imaged mouse ear blood vessels, sebaceous glands, as well as several tissue structures. We further conducted a comparative study of OCTA for rodent retinal imaging by evaluating the performance of three OCTA algorithms, namely the phase variance (PV), improved speckle contrast (ISC), and optical microangiography (OMAG). It was concluded that the OMAG algorithm provided statistically significant higher mean values of BVD and VPI compared to the ISC algorithm (0.27±0.07 vs. 0.24±0.05 for BVD; 0.09±0.04 and 0.08±0.04 for VPI), while no statistically significant difference was observed for VDI and VCI among the algorithms. Results showed that both the ISC and OMAG algorithms are more robust than PV, and they can reveal similar vasculature features. Lastly, we utilized the proposed imaging system to monitor, for the first time, the invasion process of malaria parasites in the mosquito midgut. The system shows a promising potential to detect parasite motion as well as structural changes inside the mosquito midgut. The multimodal imaging system outlined in this dissertation can be useful in a variety of applications thanks to the specific optical contrast offered by each employed modality, including retinal and brain imaging

Retinal layer segmentation in rodent OCT images: Local intensity profiles & fully convolutional neural networks

[EN] Background and Objective: Optical coherence tomography (OCT) is a useful technique to monitor retinal layer state both in humans and animal models. Automated OCT analysis in rats is of great relevance to study possible toxic effect of drugs and other treatments before human trials. In this paper, two different approaches to detect the most significant retinal layers in a rat OCT image are presented. Methods: One approach is based on a combination of local horizontal intensity profiles along with a new proposed variant of watershed transformation and the other is built upon an encoder-decoder convolutional network architecture. Results: After a wide validation, an averaged absolute distance error of 3.77 +/- 2.59 and 1.90 +/- 0.91 mu m is achieved by both approaches, respectively, on a batch of the rat OCT database. After a second test of the deep-learning-based method using an unseen batch of the database, an averaged absolute distance error of 2.67 +/- 1.25 mu m is obtained. The rat OCT database used in this paper is made publicly available to facilitate further comparisons. Conclusions: Based on the obtained results, it was demonstrated the competitiveness of the first approach since outperforms the commercial Insight image segmentation software (Phoenix Research Labs) as well as its utility to generate labelled images for validation purposes speeding significantly up the ground truth generation process. Regarding the second approach, the deep-learning-based method improves the results achieved by the more conventional method and also by other state-of-the-art techniques. In addition, it was verified that the results of the proposed network can be generalized to new rat OCT images.Animal experiment permission was granted by the Danish Animal Experimentation Council (license number: 2017-15-020101213). We gratefully acknowledge the support of NVIDIA Corporation with the donation of the Titan V GPU used for this research. This work has received funding from Horizon 2020, the European Union's Framework Programme for Research and Innovation, under grant agreement No. 732613 (GALAHAD Project), the Spanish Ministry of Economy and Competitiveness through project DPI2016-77869 and GVA through project PROMETEO/2019/109.Morales, S.; Colomer, A.; Mossi García, JM.; Del Amor, R.; Woldbye, D.; Klemp, K.; Larsen, M.... (2021). Retinal layer segmentation in rodent OCT images: Local intensity profiles & fully convolutional neural networks. Computer Methods and Programs in Biomedicine. 198:1-14. https://doi.org/10.1016/j.cmpb.2020.105788S11419

Monitoring new long-lasting intravitreal formulation for glaucoma with vitreous images using optical coherence tomography

Intravitreal injection is the gold standard therapeutic option for posterior segment patholo-gies, and long-lasting release is necessary to avoid reinjections. There is no effective intravitreal treatment for glaucoma or other optic neuropathies in daily practice, nor is there a non-invasive method to monitor drug levels in the vitreous. Here we show that a glaucoma treatment combining a hypotensive and neuroprotective intravitreal formulation (IF) of brimonidine–Laponite (BRI/LAP) can be monitored non-invasively using vitreoretinal interface imaging captured with optical coherence tomography (OCT) over 24 weeks of follow-up. Qualitative and quantitative characterisation was achieved by analysing the changes in vitreous (VIT) signal intensity, expressed as a ratio of retinal pigment epithelium (RPE) intensity. Vitreous hyperreflective aggregates mixed in the vitreous and tended to settle on the retinal surface. Relative intensity and aggregate size progressively decreased over 24 weeks in treated rat eyes as the BRI/LAP IF degraded. VIT/RPE relative intensity and total aggregate area correlated with brimonidine levels measured in the eye. The OCT-derived VIT/RPE relative intensity may be a useful and objective marker for non-invasive monitoring of BRI/LAP IF

An active contour approach for segmentation of intra-retinal layers in optical coherence tomography images

Optical Coherence Tomography (OCT) is a non-invasive, depth-resolved imaging modality that has become a prominent ophthalmic diagnostic technique. We present a novel segmentation algorithm based on Chan-Vese\u27s energy-minimizing active contours to detect intra-retinal layers in OCT images. A multi-phase framework with a circular shape prior is adopted to model the boundaries of retinal layers and estimate shape parameters using least squares. We use a contextual scheme to balance the weight of different terms in the energy functional. The results from various synthetic experiments and segmentation results on rat OCT images are presented, demonstrating the strength of our method to detect the desired layers with sufficient accuracy even in the presence of intensity inhomogeneity. Our algorithm achieved an average Dice similarity coefficient of 0.84 over all segmented layers, and of 0.94 for the combined nerve fiber layer, ganglion cell layer, and inner plexiform layer, which are critical layers for glaucomatous degeneration

EFFICACY OF INTRAVITREAL AFLIBERCEPT IN MACULAR TELANGIECTASIA TYPE 1 IS LINKED TO THE OCULAR ANGIOGENIC PROFILE.

To evaluate intravitreal aflibercept in macular telangiectasia Type 1 (MacTel 1) patients and measure their ocular angiogenic profile. Eight subjects with MacTel 1 refractory to bevacizumab, ranibizumab, or laser therapy and switched to aflibercept were included. Best-corrected visual acuity, central macular thickness, and cystic areas quantified on optical coherence tomography B-scans were assessed during 12 months. Perifoveal capillary densities were measured on optical coherence tomography angiography. Aqueous humor was sampled from six patients and eight control subjects undergoing cataract extraction. Growth factors were quantified using a multiarray immunoassay. Over 12 months, patients received 6.6 ± 1.4 (range, 5-8) intravitreal aflibercept injections. Twelve months after switching to aflibercept, best-corrected visual acuity increased by ≥5 letters in 5 of 8 patients, compared with preaflibercept levels. Mean best-corrected visual acuity improved from 79.6 (∼20/50) to 88.0 (∼20/35) Early Treatment Diabetic Retinopathy Study letters (P = 0.042), and central macular thickness decreased from 434 ± 98 μm to 293 ± 59 μm (P = 0.014). Compared with control subjects, the profile of angiogenic factors in MacTel 1 eyes revealed no difference in vascular endothelial growth factor-A levels but significantly higher levels of placental growth factor (P = 0.029), soluble vascular endothelial growth factor receptor-1 (sFlt-1; P = 0.013), vascular endothelial growth factor-D (P = 0.050), and Tie-2 (P = 0.019). Placental growth factor levels inversely correlated with both superficial and deep capillary plexus densities on optical coherence tomography angiography (P = 0.03). The clinical response to aflibercept coupled to the angiogenic profile of MacTel 1 eyes support the implication of the placental growth factor/Flt-1 pathway in MacTel 1

Visible Optical Coherence Tomography based Multimodal Imaging for Quantification of Retinal Lipofuscin

Retinal degeneration is the leading cause of irreversible low vision and blindness in the world, that describes conditions characterized by progressive loss of photoreceptors. Retinal Pigment Epithelium (RPE) is located under photoreceptors’ outer segments and plays an important role in the maintenance of photoreceptors by completing the visual cycle and phagocytosis of shed photoreceptor outer segments. Lipofuscin, a byproduct of the visual cycle, is a nondegradable compound that accumulates in the RPE cells and eventually damages the RPE cells and inevitably causes photoreceptor degeneration. Lipofuscin is the major cause of fundus fluorescence that can be detected by Fundus Autofluorescent (FAF) imaging systems. Reliable and quantified FAF values are necessary for lipofuscin quantification which can be a significant tool in the diagnosis of retinal degenerative disease in early stages and provide a better opportunity for treatment before the loss of vision stage. However, FAF is attenuated by the ocular media prior to the RPE, including cornea, lens, vitreous body, retinal layers in front of the RPE, and the melanin granules within the RPE cells that migrate to the apical region upon light exposure. This attenuation varies among people and for an individual over time and cannot be measured directly, thus hurdles measurement of the true FAF values. Further, differences in acquisition systems such as illumination power and detector sensitivity, directly affect the measured FAF. This issue has been addressed by implementing a reference target in the FAF imaging system. Normalizing the FAF signal to that of the target eliminates the dependency on the acquisition parameters. However, the issue of pre-RPE and RPE melanin attenuation remains unresolved. Further, the fluorescence characteristics of the commercially available fluorescent reference are quite different than retinal lipofuscin that challenges the quantification of the absolute amount of lipofuscin in the RPE. In this dissertation, we propose a new multimodal imaging system based on visible-light optical coherence tomography (VIS-OCT) that provides a three-dimensional image. The technology simultaneously acquires VIS-OCT and FAF with a single broadband visible light source. Since both images are originated from the same group of photons and travel through the same ocular media at the same time, the attenuation factor is similar in both modalities. Therefore, by normalizing FAF by VIS-OCT of the RPE layer, the attenuation of the pre_RPE media can be eliminated. Further, we implemented two reference targets to quantify VIS-OCT and FAF and eliminate the dependency on acquisition parameters. These references were later substituted by a single customized reference that consists of the major lipofuscin fluorophore, called A2E. The quantitative imaging independent of system fluctuation, and attenuation of pre-RPE and RPE melanin was successfully tested on retinal simulating phantoms, in vivo on the animal retina, and human subjects. The in vivo quantification in small animals linearly correlates with A2E content measured by mass spectrometry. Quantitative imaging of human retinas is consistent with the linear accumulation of lipofuscin with age. The VIS-OCT-FAF has the potential for clinical diagnosis

Preventing corneal calcification associated with xylazine for longitudinal optical coherence tomography in young rodents

PURPOSE. Spectral-domain optical coherence tomography (SD-OCT) is widely used in clinical ophthalmology and recently gained popularity in laboratory research involving small rodents. Its noninvasive nature allows repeated measurements, thereby decreasing the number of animals required. However, when used at a conventional dosage, xylazine (an a2- adrenoceptor) can cause irreversible corneal calcification, especially among young rodents. In the present study, we test whether corneal calcification associated with xylazine is mediated by the a2-adrenoceptor. METHODS. Our study tested Sprague-Dawley rats, Long-Evans rats, and CD-1 mice (postnatal day [P]14). Retinal images were captured by SD-OCT. Quantitative PCR (qPCR) was used to study gene expression, whereas receptor localization was examined by immunofluorescent staining followed by confocal microscopy. Calcium deposits were detected via von Kossa staining. RESULTS. When used at dosages appropriate for adult animals, ketamine-xylazine anesthetics led to a high rate of respiratory failure, increased apoptotic activity in the corneal epithelium, and irreversible corneal calcification in P14 rat pups. Meanwhile, OCT image quality decreased drastically as a result of corneal calcification among animals recovering from anesthesia. a2-Adrenoceptor subtypes were highly expressed on P14, in line with rodents’ age-specific sensitivity to xylazine. Clonidine, a potent a2-adrenoceptor agonist, dosedependently induced corneal calcification, which could be prevented by an a2-adrenoceptor antagonist. CONCLUSIONS. These data suggest that a2-adrenoceptors contribute to corneal calcification in young rodents. Therefore, we developed a suitable OCT imaging protocol for this cohort, including a carefully tailored ketamine-xylazine dosage (60 mg/kg and 2.5 kg/mg, respectively)

Automated choroidal segmentation of 1060 nm OCT in healthy and pathologic eyes using a statistical model

A two stage statistical model based on texture and shape for fully automatic choroidal segmentation of normal and pathologic eyes obtained by a 1060 nm optical coherence tomography (OCT) system is developed. A novel dynamic programming approach is implemented to determine location of the retinal pigment epithelium/ Bruch’s membrane /choriocapillaris (RBC) boundary. The choroid–sclera interface (CSI) is segmented using a statistical model. The algorithm is robust even in presence of speckle noise, low signal (thick choroid), retinal pigment epithelium (RPE) detachments and atrophy, drusen, shadowing and other artifacts. Evaluation against a set of 871 manually segmented cross-sectional scans from 12 eyes achieves an average error rate of 13%, computed per tomogram as a ratio of incorrectly classified pixels and the total layer surface. For the first time a fully automatic choroidal segmentation algorithm is successfully applied to a wide range of clinical volumetric OCT data

Quantitative Optical Studies of Oxidative Stress in Rodent Models of Eye and Lung Injuries

Optical imaging techniques have emerged as essential tools for reliable assessment of organ structure, biochemistry, and metabolic function. The recognition of metabolic markers for disease diagnosis has rekindled significant interest in the development of optical methods to measure the metabolism of the organ. The objective of my research was to employ optical imaging tools and to implement signal and image processing techniques capable of quantifying cellular metabolism for the diagnosis of diseases in human organs such as eyes and lungs. To accomplish this goal, three different tools, cryoimager, fluorescent microscope, and optical coherence tomography system were utilized to study the physiological metabolic markers and early structural changes due to injury in vitro, ex vivo, and at cryogenic temperatures. Cryogenic studies of eye injuries in animal models were performed using a fluorescence cryoimager to monitor two endogenous mitochondrial fluorophores, NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). The mitochondrial redox ratio (NADH/ FAD), which is correlated with oxidative stress level, is an optical biomarker. The spatial distribution of mitochondrial redox ratio in injured eyes with different durations of the disease was delineated. This spatiotemporal information was helpful to investigate the heterogeneity of the ocular oxidative stress in the eyes during diseases and its association with retinopathy. To study the metabolism of the eye tissue, the retinal layer was targeted, which required high resolution imaging of the eye as well as developing a segmentation algorithm to quantitatively monitor and measure the metabolic redox state of the retina. To achieve a high signal to noise ratio in fluorescence image acquisition, the imaging was performed at cryogenic temperatures, which increased the quantum yield of the intrinsic fluorophores. Microscopy studies of cells were accomplished by using an inverted fluorescence microscope. Fixed slides of the retina tissue as well as exogenous fluorophores in live lung cells were imaged using fluorescent and time-lapse microscopy. Image processing techniques were developed to quantify subtle changes in the morphological parameters of the retinal vasculature network for the early detection of the injury. This implemented image cytometry tool was capable of segmenting vascular cells, and calculating vasculature features including: area, caliber, branch points, fractal dimension, and acellular capillaries, and classifying the healthy and injured retinas. Using time-lapse microscopy, the dynamics of cellular ROS (Reactive Oxygen Species) concentration was quantified and modeled in ROS-mediated lung injuries. A new methodology and an experimental protocol were designed to quantify changes of oxidative stress in different stress conditions and to localize the site of ROS in an uncoupled state of pulmonary artery endothelial cells (PAECs). Ex vivo studies of lung were conducted using a spectral-domain optical coherence tomography (SD-OCT) system and 3D scanned images of the lung were acquired. An image segmentation algorithm was developed to study the dynamics of structural changes in the lung alveoli in real time. Quantifying the structural dynamics provided information to diagnose pulmonary diseases and to evaluate the severity of the lung injury. The implemented software was able to quantify and present the changes in alveoli compliance in lung injury models, including edema. In conclusion, optical instrumentation, combined with signal and image processing techniques, provides quantitative physiological and structural information reflecting disease progression due to oxidative stress. This tool provides a unique capability to identify early points of intervention, which play a vital role in the early detection of eye and lung injuries. The future goal of this research is to translate optical imaging to clinical settings, and to transfer the instruments developed for animal models to the bedside for patient diagnosis