Clinical Optical Coherence Tomography Angiography Registration and Analysis

Transcription

1 Clinical Optical Coherence Tomography Angiography Registration and Analysis by Morgan Lindsay Heisler B.A.Sc. (Hons.), Simon Fraser University, 2015 Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Applied Science in the School of Engineering Science Faculty of Applied Sciences Morgan Lindsay Heisler SIMON FRASER UNIVERSITY Spring 2017 Copyright in this work rests with the author. Please ensure that any reproduction or re-use is done in accordance with the relevant national copyright legislation.

2 Approval Name: Degree: Title: Morgan Lindsay Heisler Master of Applied Science Clinical Optical Coherence Tomography Angiography Registration and Analysis Examining Committee: Chair: Dr. Ash M. Parameswaran, P. Eng. Professor Dr. Marinko V. Sarunic, P. Eng. Senior Supervisor Professor Dr. Mirza Faisal Beg, P. Eng. Supervisor Professor Dr. Yifan Jian Supervisor Adjunct Professor Dr. Paul J. Mackenzie Supervisor Clinical Assistant Professor Ophthalmology and Visual Sciences University of British Columbia Dr. Eduardo V. Navajas External Examiner Clinical Assistant Professor Ophthalmology and Visual Sciences University of British Columbia Date Defended/Approved: April 20th, 2017 ii

3 Ethics Statement iii

4 Abstract Optical Coherence Tomography Angiography (OCT-A) is an emerging imaging modality with which the retinal circulation can be visualized by computing the decorrelation signal on a pixel-by-pixel basis. This non-invasive, in vivo visualization of the retinal microvasculature can be instrumental in studying the onset and development of retinal vascular diseases. Quantitative measurements, such as capillary density, can be used to stratify the risk of disease progression, visual loss, and also for monitoring the course of disease. Due to projection artifact and poor contrast, it is often difficult to trace individual vessels when only one en face image is visualized. Averaging of up to 10 serially acquired OCT-A images with parallel strip-wise microsaccadic noise removal and localized nonrigid registration is presented. Additionally, the use of a deep learning method for the quantification of Foveal Avascular Zone (FAZ) parameters and perifoveal capillary density of prototype and commercial OCT-A platforms in both healthy and diabetic eyes is evaluated. Keywords: optical coherence tomography; ophthalmology; image processing; registration; retina; angiography iv

5 Acknowledgements I would like to express my deepest gratitude to my senior supervisor, Dr. Marinko V. Sarunic, whose expertise, understanding, and patience added considerably to my graduate experience. I appreciate his confidence and trust in my abilities while pushing me outside my academic comfort zone. I would also like to sincerely thank Dr. Mirza Faisal Beg for all of his expertise in medical image processing which was crucial to the completion of this research. His passion and enthusiasm for the research made every interaction an enjoyable experience. I would also like to acknowledge the medical professionals who have helped shape my graduate career. Dr. Paul J. Mackenzie and Dr. Eduardo V. Navajas both took time out of their busy clinical practices to share invaluable medical expertise and direction for these projects. A special thanks also goes out to Dr. Zaid Mammo for his dedication to the research. His patience and willingness to explain concepts that must have seemed very basic to a resident of his caliber were so appreciated. Additionally, I would like to thank the international collaborators who have helped with this research. Dr. Dao-Yi Yu and Dr. Chandrakumar Balaratnasingam were extremely welcoming and supportive during my time in Perth as well as everyone else in the Pathology and Physiology Department at Lions Eye Institute. Also, a huge thank you to Dr. Pavle Prentašić for exposing me to interesting world of machine learning. I am very grateful to be part of the Biomedical Optics Research Group (BORG) at SFU. I am especially grateful to BORG members Dr. MyeongJin Ju, Dr. Sieun Lee and Dr. Yifan Jian for their mentorship in these past few years. They have demonstrated some of the traits that I can only strive for in future endeavours: Dr. Ju s dedicated work ethic, Dr. Lee s eye for detail, and Dr. Jian s willingness to make the work environment as enjoyable as possible. Lastly, I would like to thank my family for their support and encouragement in all my endeavours. I couldn t have done it without you. v

6 Table of Contents Approval... ii Ethics Statement... iii Abstract... iv Acknowledgements... v Table of Contents... vi List of Tables... viii List of Figures... ix List of Acronyms... xi Chapter 1. Introduction Eye Anatomy Diabetic Retinopathy Glaucoma Ophthalmic Imaging Modalities Fluorescein Angiography Optical Coherence Tomography Angiography Contributions Outline of Thesis... 8 Chapter 2. Research Motivation Methods Visual Fields, Disc Photographs, Peripapillary Optical Coherence Tomography Optical Coherence Tomography Angiography Instrumentation Processing of OCT-A Images Image Acquisition and Quantification Results Discussion Summary Chapter 3. OCT-A Image Strip-based Registration Methods Optical Coherence Tomography Instrumentation En face Angiogram Extraction Angiogram Registration Microsaccade Free Strip Generation Strip-based Affine Registration Strip-based Non-Rigid Registration Validation Results Discussion Summary vi

7 Chapter 4. Automated Quantification Methods Inclusion Criteria Optical Coherence Tomography Instrumentation Imaging Protocols Processing of OCT-A Images Manual Tracing Methods Algorithm Training Methods Segmentation Performance Analysis Clinical Outcome Measures Results Deep Neural Network Algorithm Performance Clinical Outcome Measures Discussion Summary Chapter 5. Future Work D Registered OCTA Volumes Registration of Photoreceptor Images Apply the DNN Framework to Averaged Images References Appendix. Further Examples of Averaged OCT-A Images vii

8 List of Tables Table 1. Clinical Outcome Measures viii

9 List of Figures Figure 1.1 Representative images of the Indian Ocean seen by someone with A) normal vision, B) diabetic retinopathy, and C) glaucoma Figure 1.2 Eye diagram [credit: National Eye Institute, National Institutes of Health]. 2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 2.1 Figure 2.2 Figure 3.1 Figure 3.2 A portion of the human retina. Transverse histological retinal section was stained with toluidine blue (A), and a B-scan image was acquired using OCT (B) to illustrate the various retinal layers at the eccentricity located 3 mm superior to the optic disk. Colored dashed lines demarcate the retinal layers. Orange dashed lines indicate NFL; red dashed lines, RGC capillary network; yellow dashed lines, capillary network at IPL/sINL border; green dashed lines, capillary network at the dinl/opl border. Scale bar: 50 μm. Image from [1] Fundus photograph showing fluorescein imaging of the major arteries and veins in a human left eye An example of OCTA images processed using the first version of the processing pipeline (left), and the current version of the pipeline (right)... 7 Manual tracing techniques for quantifying radial peripapillary capillary (RPC) density. Radial peripapillary capillaries are seen in the speckle variance OCT-A image of a normal eye (Top). Manual tracing of RPCs (Center; red) were performed, the results of which were used to express the density of RPCs as a percentage of the total tissue area (Bottom). Note that large vessels were excluded from the tracing. Scale bar = 300 μm Structural changes to radial peripapillary capillaries (RPCs) in unilateral glaucoma. The right optic disc (Top row, first image) demonstrates a myopic tilt however the automated Humphrey visual field test (Second row, first image) appears normal. Speckle variance OCT-A images of RPCs (Third row, first image) and the deep capillary plexus (Fourth row, first image) in the superotemporal peripapillary region are within the normal range. The left glaucomatous eye also demonstrates tilting (Top row, second image) but an inferior field defect is seen on visual field examination (Second row, second image). There is loss of RPCs in the superotemporal peripapillary region (Third row, second image) as seen on the speckle variance OCT-A image. The deeper capillary plexus at sites of RPC loss however appears normal and comparable in morphology to the fellow eye (Fourth row, second image). Projection artifacts from the large retinal vessels within the inner retina could be seen in the deeper capillary plexus images in both eyes (Fourth row, first and second image). Scale bar = 300μm Overview of the strip-based registration algorithm for multiple serially acquired OCT-A images. Representative images are used to demonstrate the algorithm in Figure Demonstration of the image stripping, coarse translation, affine registration and non-rigid registration steps of the proposed algorithm. The template image (green) and registered strip (magenta) are shown as composite images where white regions indicate where the two images ix

10 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 4.1 Figure 4.2 Figure 4.3 Figure 5.1 have the same intensities. The areas under the red, orange and yellow boxes are further explored in Figure Comparison of three different strips registered to the same template image using (a) coarse translation, (b) affine registration, and (c) non-rigid registration. The template image (green) and registered strip (magenta) are shown as composite images where white regions indicate where the two images have the same intensities Template image, mean, and median averaged images (all retinal layers, superficial and deep plexus) for Subject 3 OD, a healthy male subject, 29 years of age Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a subject with diabetic retinopathy.. 28 SSIM values for incremental averaged images for all eyes for the all retinal layers, superficial layers and deep layers An example image from both the prototype and commercial systems with their corresponding manual and automated segmentations. As some data within the commercial dataset contained an icon in the lower left corner a mask was applied and can be seen in the lower left corner of the automated segmentation result FAZ perimeter (yellow), maximum diameter (green) and minimum diameter (red) shown for example healthy and diabetic data from both systems using both manually and automated segmentations. As some data within the commercial dataset contained an icon in the lower left corner a mask was applied and can be seen in the lower left corner of the commercial automated segmentation results (white) and the lower left corner in the manual segmentation of the commercial diabetic image (black) Examples of low quality input data and the automated segmentation. Due to the low signal-to-noise ratio within the FAZ, some areas were erroneously segmented. Additionally, a horizontal motion artefact can be seen cutting through the FAZ which was also incorrectly segmented in areas Two AO-OCT images of photoreceptors acquired from the same patient in a similar area. Scale bar is 50μm x

11 List of Acronyms CNR DNN DR FA FAZ FWHM ICC ILM INL IOP IPL OCT OCT-A ONH ONL OPL RPC SIFT SNR SSADA SSIM VF Contrast to Noise Ratio Deep Neural Networks Diabetic Retinopathy Fluorescein Angiography Foveal Avascular Zone Full Width Half Maximum Intraclass Correlation Coefficients Inner Limiting Membrane Inner Nuclear Layer Intra-Ocular Pressure Inner Plexiform Layer Optical Coherence Tomography Optical Coherence Tomography Angiography Optic Nerve Head Outer Nuclear Layer Outer Plexiform Layer Radial Peripapillary Capillaries Scale Invariant Feature Transform Signal to Noise Ratio Split-spectrum amplitude-decorrelation angiography Structural Similarity Index Measure Visual Field xi

12 Chapter 1. Introduction Vision is one of the five senses that many people take for granted every day. Retinal diseases are one of the leading causes to affect the light sensitive tissue at the back of the eye, affecting the ability to detect light. Two of the most common eye diseases that cause blindness are diabetic retinopathy and glaucoma. Simulated examples of the impacts on vision from these diseases are shown in Figure 1.1. Diabetic Retinopathy (DR) causes a partial blurring or patchy loss of vision as shown in Figure 1.1B. Patients with glaucoma, on the other hand, may experience loss of peripheral vision, called tunnel vision, as depicted in Figure 1.1C. As our population ages, the number of people affected by these diseases is expected to rise significantly. The National Eye Institute estimates that number of people who will have DR will nearly double from 7.7 million to 14.6 million and the number of people with glaucoma will more than double from 2.7 million to 6.3 million from 2010 to 2050 in the United States. This increases the need for better diagnostic tools which enable ophthalmologists to detect these diseases earlier and with better confidence. Figure 1.1 Representative images of the Indian Ocean seen by someone with A) normal vision, B) diabetic retinopathy, and C) glaucoma. Over the past decade, the development of visible and near-infrared retinal imaging technology has grown rapidly. One of the dominant imaging modalities is Fourier Domain Optical Coherence Tomography (FDOCT), which has revolutionized clinical diagnostic ophthalmic imaging. FDOCT provides a detailed volumetric view of the retina for clinicians to identify the structural hallmarks of diseases such as DR and 1

13 glaucoma. FDOCT images are used to assess the need for treatments (surgical, intravitreal injection, laser, etc.) and afterwards to evaluate the results and monitor changes. In the remainder of this chapter, an overview of the eye anatomy, with a focus on the retina is presented. This is followed by a detailed description of DR and glaucoma. The imaging technologies that are specifically used by clinicians for visualizing the retinal vasculature are described. The chapter concludes with an overview of the rest of this thesis, and my contributions towards the development of tools for the betterment of Optical Coherence Tomography Angiography (OCT-A) images Eye Anatomy The eye is a complex organ that allows us to perceive and convert light to electrical signals that the brain can interpret. Figure 1.2 presents a simple schematic of a human eye. Figure 1.2 Eye diagram [credit: National Eye Institute, National Institutes of Health] 2

14 Briefly, a collimated beam incident on the eye is focused by the cornea and lens onto the retina. The eye is roughly 25mm in diameter, and the retina, located at the back of the eye contains cell layers that detect light, perform some processing on the information, and transmit electrical signals to the brain via the neurons of the optic nerve. The rest of the report will focus mainly on the retina, a cross sectional diagram of which is shown in Figure 1.3. Figure 1.3 A portion of the human retina. Transverse histological retinal section was stained with toluidine blue (A), and a B-scan image was acquired using OCT (B) to illustrate the various retinal layers at the eccentricity located 3 mm superior to the optic disk. Colored dashed lines demarcate the retinal layers. Orange dashed lines indicate NFL; red dashed lines, RGC capillary network; yellow dashed lines, capillary network at IPL/sINL border; green dashed lines, capillary network at the dinl/opl border. Scale bar: 50 μm. Image from [1]. There are two sources of blood supply to the human retina: the central retinal artery (CRA) and the choroidal blood vessels. The inner retina is bounded by the nerve fibre layer (NFL) anteriorly and the inner nuclear layer (INL) posteriorly, and it gets nourished by blood that travels through the CRA from the optic nerve head (ONH). The choroidal blood vessels feed the outer retina, particularly the photoreceptors. The CRA follows the patterns as shown in Figure 1.4, where the vessels radiate outward from the ONH and curve towards and around the fovea. These vessels supply three layers of capillary networks which are the radial peripapillary capillaries (RPCs), the inner and outer layer of capillaries. The RPCs are the most superficial layer of capillaries lying in the inner part of NFL, and feed the superficial nerve fibres surrounding the ONH. The inner capillaries lie in the GCLs under and parallel to the RPCs. The outer 3

15 capillary network runs from the inner plexiform layer (IPL) to the outer plexiform layer (OPL) through the inner nuclear layer (INL) [8]. Figure 1.4 Fundus photograph showing fluorescein imaging of the major arteries and veins in a human left eye Diabetic Retinopathy Diabetic retinopathy (DR) is the most prevalent retinal vascular disease worldwide, affecting a third of people with diabetes [2]. It is a leading cause of adult blindness, responsible for 15-17% of cases of blindness in the western world [3]. The pathophysiology of DR is closely related to its deleterious effect on the inner retinal microcirculation which includes altered vascular permeability and capillary bed closure [4], [5]. Retinal ischemia secondary to capillary non-perfusion has been observed in the early stages of diabetic retinopathy and has been correlated to disease severity and progression [6]. Findings such as decreasing macular capillary density and enlargement of the perifoveal zone [7] have been correlated with the severity of vision loss [8], [9]. Thus, early detection and reliable quantification of these microvascular changes may play a role in predicting visual morbidity and improve the management of DR. 4

16 1.3. Glaucoma Glaucoma is a leading cause of irreversible blindness worldwide[10] and the second most common cause of blindness in the developed world [11]. The pathophysiology of glaucoma is complex and characterized by the time-dependent loss of retinal ganglion cells (RGCs) and their accompanying axons [12]. Indices that are currently used to quantify and evaluate progression of glaucomatous optic neuropathy include visual field testing, nerve fibre layer (NFL), optic nerve head, ganglion cell layer with inner plexiform layer (GCIPL) and ganglion cell complex parameters analysis, and, recently, measurement of lamina cribrosa thickness [13]. The RPCs represent a unique capillary plexus within the inner aspect of the NFL. They are largely restricted to the posterior pole of the human retina along specific retinal eccentricities surrounding the optic nerve. Morphologically, this capillary network displays minimal inter-capillary anastomosis and show a linear course in keeping with the NFL distribution. The anatomical distribution and unique morphological characteristics help to distinguish the RPCs from other capillary plexuses within the retinal microcirculation [12], [14]. The nutritional demands of RGC axons are likely to be partially satisfied to a large extent satisfied by radial peripapillary capillaries (RPCs) [15] and structural changes to RPCs have been implicated in the pathogenesis of glaucoma [16]. Despite the evidence that RPCs are critically related to RGC function [17] [19], the morphological characteristics of RPCs are not routinely used in clinical practice to evaluate glaucomatous progression. This may be because RPCs are not reliably visualized with fluorescein angiography (FA) [20] which is the mainstay imaging modality for clinically evaluating the retinal circulation Ophthalmic Imaging Modalities The unique properties of the eye make it suitable for non-invasive optical imaging. Imaging blood flow is very important because abnormal circulation is the leading cause of irreversible blindness in diseases such as DR. Here we focus on fluorescein angiography (FA) and Optical Coherence Tomography Angiography (OCT- A). 5

17 Fluorescein Angiography For the past 30 years, fluorescein angiography (FA) has been the gold standard modality for assessing retinal vascular diseases [17] but vessel leakage and excessive choroidal fluorescence affects the ability of FA to visualize the retinal microcirculation. FA is an invasive procedure that requires venipuncture and the administration of exogenous contrast agents. The injected dye can cause nausea, vomiting, skin discolouration, pruiritis and in rare cases death and anaphylaxis [46]. Moreover, these techniques only provide 2D information (en face views of the vasculature). Therefore, there is a clinical demand for a non-invasive approach to provide visualization of the microvasculature within the retina layers Optical Coherence Tomography Angiography Optical Coherence Tomography Angiography (OCT-A) is an emerging imaging modality with which the retinal circulation can be visualized by computing the decorrelation signal on a pixel-by-pixel basis. Variants of OCT-A methods have been described in recent review articles [21] [24]. The speckle variance approach to OCT-A has been evaluated against standard invasive techniques such as Fluorescein Angiography (FA) [25], [26], in which only the superficial capillaries can be distinguished due to excessive choroidal fluorescence[27], and ex vivo histological analyses [1], [26], [28], [29] Contributions At the early stage of my graduate research, my work was mainly focused on data acquisition, where I became familiar with the potential uses and limitations of OCT Angiography. I was able to acquire and help analyze OCT-A data of the Optic Nerve Head (ONH) looking closely at the RPCs which contributed to a published paper [30]. Through analyzing this data, I took the first version of the OCT-A processing pipeline (which was on a single computer in the BORG lab at SFU and required the use of a special version of the custom OCTViewer acquisition software) and adapted it to work on a computer cluster server so that any team member could process the data. Although the first version of the code was useful for research, the inability to parallelize the processing made it too slow to produce a clinical output in one day. Additional layers 6

18 were also added to the graph cut segmentation codes and additional filters were added to emphasize the details of the fine microvasculature. This can be seen in Figure 1.5, in which the capillaries have a higher contrast (qualitatively) with the updated processing methods. Figure 1.5 An example of OCTA images processed using the first version of the processing pipeline (left), and the current version of the pipeline (right). By working on this study, two needs became apparent: 1) better quality en face OCT-A angiograms, and 2) a more automated process for quantifying the data was needed for accurate clinical diagnosis. Through working with an exchange PhD student, a process for segmenting the retinal vasculature in our OCT-A data using Deep Neural Networks (DNNs) was developed which led to two co-first authored papers [31],[32]. My main first-author paper [33] demonstrated an automated method for registration and averaging of serially acquired OCT-A images. The improved visualization of the capillaries will hopefully enable more robust quantification and study of minute changes in retinal microvasculature in the future. 7

19 Although my main contributions pertain to our OCT-A studies [26], [30] [34], I was also able to contribute to some of the adaptive optics [35],[36] anterior segment [37] and morphological analysis [38],[39] work in a lesser capacity Outline of Thesis The remainder of this thesis is organized as follows. In Chapter 2, motivation for the research is presented in the form of a clinical study which utilizes OCT-A to image the RPCs in focal glaucoma. Chapter 3 details an image processing pipeline for the registration and averaging of serially acquired OCT-A images. Chapter 4 investigates the ability of machine learning for automated analysis of OCT-A data. Lastly, the thesis ends with a summary and future work. 8

20 Chapter 2. Research Motivation Previous studies evaluated the morphological characteristics of the foveal [26], perifoveal [28] and peripapillary capillary [29] networks using OCT-A [25] and showed that the topological and quantitative characteristics of these networks, as seen on OCT- A, are comparable to histologic representation. This chapter utilizes OCT-A to quantitatively evaluate RPCs in glaucoma, glaucoma suspects, and normal eyes which provides clinical motivation for the research presented in this thesis Methods The study was designed as a prospective observational case series. All subject recruitment and imaging took place at the Eye Care Centre at Vancouver General Hospital. The study protocol including subject recruitment and imaging was approved prospectively by the Research Ethics Boards at the University of British Columbia and Vancouver General Hospital. The study was performed in accordance and adhered with the tenets of the Declaration of Helsinki. Written informed consent was obtained from all subjects Visual Fields, Disc Photographs, Peripapillary Optical Coherence Tomography Visual fields were acquired using the Humphrey Field Analyzer II (Carl Zeiss Meditec, Dublin, CA). Refractive error was corrected during testing. Stereoscopic photos around the optic discs were obtained for each participant using a fundus camera (TRC- 50DX; Topcon, Japan) with 5.0-megapixel resolution. Peripapillary assessment of the NFL was done using the standard peripapillary protocol using SD-OCT (Spectralis, Heidelberg Engineering, Germany). All ancillary testing were acquired within six months of OCT-A imaging. 9

21 Optical Coherence Tomography Angiography Instrumentation Speckle variance OCT-A images and simultaneous, co-registered regular structural OCT images were acquired from a GPU-accelerated OCT clinical prototype. The details of the acquisition system have previously been published [25]. The OCT system used a 1060nm swept source (Axsun Inc.) with 100 khz A-scan rate and a fullwidth half-maximum bandwidth of 61.5nm which corresponded to a coherence length of ~6μm in tissue. The size of the focal waist on the retina was estimated using the Gullstrand-LeGrand model of the human eye to be ω μm (calculated using Gaussian optics) corresponding to a lateral FWHM of ~8.6 μm. For the angiogram, the speckle variance calculation [40] was used N N 1 1 sv ( I I ) jk ijk ijk N i 1 N i 1 2, (1) where i, j, and k are the indices of the frame, width, and axial position of the B-scan respectively, I is the intensity at the index and N is the number of repeat acquisitions per BM-scan (N=3). Processing of the OCT intensity image data and en face visualization of the retinal microvasculature was performed in real time using our open source code for alignment and quality control purposes [41], [42]. The scan area was sampled in a 300x300(x3) grid with a ~2x2mm field of view in 3.15 seconds. Scan dimensions were calibrated based on the eye length of each participant, measured using the IOL Master 500 (Carl Zeiss Meditec Inc., Dublin, California, USA) Processing of OCT-A Images Post-processing of the raw intensity data was performed to segment the retinal layers and extract optimal quality images of the retinal microvasculature. Coarse axial motion artifact was corrected using cross-correlation between adjacent frames. Subpixel registration was performed on each set of corresponding B-scans before creating the speckle variance B-mode scan. Before layer segmentation, three-dimensional bounded variance smoothing was applied to the motion corrected intensity images in order to reduce the effect of speckle while preserving and enhancing edges. The inner limiting membrane, posterior boundary of the NFL, inner nuclear layer and outer nuclear 10

22 layer were segmented automatically in 3D using a graph-cut algorithm [43]. The automated segmentation was examined and corrected by a trained grader using Amira (version 5.1; Visage Imaging, San Diego, CA, USA). The OCT-A image within each layer was summed in the axial direction to produce a projected en face image. En face images were notch filtered and contrast-adjusted using adaptive histogram equalization. In an effort to eliminate the bias of large blood vessels from the NFL thickness measurement, the images were cropped to an area of ~1x1mm to remove the majority of the large blood vessels present. Furthermore, prior to performing capillary density comparisons between glaucoma, glaucoma suspect and normal control subjects the images were again cropped to an area of 636.5x636.5μm Image Acquisition and Quantification Group A consisted of subjects with unilateral glaucoma, Group B consisted of glaucoma suspects, and Group C consisted of healthy subjects. The average age of subjects (range and median) of Groups A, B and C were 58.00±18.60 (26-72: 66) years, 44.67±23.50 years (21-68: 45) and 41.13±13.51 years (27-60: 36), respectively (P=0.25). The male: female ratio of subjects in Group A, B and C were 3:2, 3:0 and 4:3, respectively. The average intraocular pressure of subjects (range and median) of the glaucoma eyes and fellow eyes in Group A and Group B were ± 4.21 mmhg (5-15: 12), ± 3.21 mmhg (10-17: 14) and ± 2.04 mmhg (14-19: 16), respectively. Our previously published manual tracing technique was used to quantify RPC density as shown in Figure 2.14 All manual tracings were performed by ZM in a nonblinded fashion. Manual tracing was performed using the GNU Image Manipulation Program Version Care was taken to trace RPCs, and all large vessels originating from the disc were segmented separately and excluded. Capillary density was measured in the segmented image using MATLAB. The proportion of the image occupied by retinal vessels was expressed as a percentage, and the unit of measurement was calculated as the percentage retinal area occupied by capillary plexus. As speckle variance images are derived from the structural intensity scans, quantitative structural information such as the NFL thickness can be extracted from the exact same location as capillary density. The NFL thickness was measured and averaged across the cropped volumetric ~1x1mm scan of the region of interest. Where 11

23 reported, the density of the RPCs was calculated as the fraction of pixels identified as belonging to a vessel versus the total number of pixels in an image. To determine the reproducibility of these measurements, three images from Group A, B and C were manually traced and quantified on two separate occasions in a blinded fashion, each at least 3 months apart, by the same rater (ZM). To facilitate the qualitative comparisons of the deep capillary plexus, careful manual segmentation of the retinal layers was performed to minimize projection artifacts. In addition, manually scrolling through the entire OCT volume helped to identify and exclude any residual projection artifacts from the overlying large superficial vessels in the qualitative comparison. Figure 2.1 Manual tracing techniques for quantifying radial peripapillary capillary (RPC) density. Radial peripapillary capillaries are seen in the speckle variance OCT-A image of a normal eye (Top). Manual tracing of RPCs (Center; red) were performed, the results of which were used to express the density of RPCs as a percentage of the total tissue area (Bottom). Note that large vessels were excluded from the tracing. Scale bar = 300 μm. 12

24 2.2. Results In the normal control group, the RPCs followed a very similar trajectory to the RGC axons in the NFL and demonstrated a linear course with minimal anastomoses. Decreased density of RPCs was observed within regions of optic disc neural rim loss in glaucomatous eyes. In glaucomatous eyes, RPCs maintained a linear trajectory, however a patchy or diffuse loss of RPCs was observed within regions of NFL thinning. The density and morphologic characteristics of deeper capillary networks, beyond the outer margins of the NFL, at sites of RPC loss appeared normal in glaucomatous eyes (Figure 2.2). 13

25 Figure 2.2 Structural changes to radial peripapillary capillaries (RPCs) in unilateral glaucoma. The right optic disc (Top row, first image) demonstrates a myopic tilt however the automated Humphrey visual field test (Second row, first image) appears normal. Speckle variance OCT-A images of RPCs (Third row, first image) and the deep capillary plexus (Fourth row, first image) in the superotemporal peripapillary region are within the normal range. The left glaucomatous eye also demonstrates tilting (Top row, second image) but an inferior field defect is seen on visual field examination (Second row, second image). There is loss of RPCs in the superotemporal peripapillary region (Third row, second image) as seen on the speckle variance OCT-A image. The deeper capillary plexus at sites of RPC loss however appears normal and comparable in morphology to the fellow eye (Fourth row, second image). Projection artifacts from the large retinal vessels within the inner retina could be seen in the deeper capillary plexus images in both eyes (Fourth row, first and second image). Scale bar = 300μm. 14

26 2.3. Discussion Radial peripapillary capillaries comprise a unique vascular plexus that is predominantly found in the posterior pole of primates with a typical macula. The metabolic demands of peripapillary RGC axons are likely to be mainly nourished by the RPCs. There is also evidence to demonstrate an association between RPC loss and NFL changes in chronic glaucoma. Although clinical imaging of RPCs may be a potentially useful way for evaluating and monitoring RGC axonal disease it is not routinely used in clinical practice due to the difficulties associated with visualizing this circulation using FA. Our previous study showed that quantitative analysis of retinal capillary detail could only be performed in 30% of FA images acquired from normal subjects with clear ocular media. The major limiting factor that precludes clear visualization of retinal capillaries on FA is fluorescence from the choroidal circulation. Our recent studies have quantified the morphological characteristics of retinal capillary networks as seen OCT-A and have shown that it is comparable to histological representation thus suggesting that OCT-A techniques may be useful for evaluating the structural characteristics of retinal capillary networks. Optical coherence tomography angiography overcomes some of the limitations of FA as it is a label-free technique that permits non-invasive, depth-resolved evaluation of retinal capillary networks [22]. As shown in Figure 2.2, RPC loss was identified in the NFL in glaucomatous eyes while the deeper capillary networks appeared structurally normal. Manual tracing techniques were used to calculate RPC density and in order for this technique to have broad clinical utility an automated method for determining RPC density will be required. Skeletonization algorithms and binary image analysis techniques may potentially overcome this limitation. All manual tracings were performed by ZM in a non-blinded fashion to minimize grader bias, all tracings were collectively reviewed and approved by ZM, MH and CB prior to analysis. Projection artifacts could have affected our qualitative comparison of the deep capillary networks between the study groups. As outlined in the methods section, a number of manual steps were taken to minimize the potential deleterious effects of projection artifacts from overlying vessels. This topic is receiving increased attention in the OCTA literature, automated methods for removing projection artifacts are still in the development phase. 15

27 2.4. Summary In this chapter, OCT-A was used to help answer a clinical question proving the clinical utility of this technology; however, there were also several limitations noted which could be improved upon specifically the low image quality and use of manual quantification. In the next chapter, a pipeline for improving the visible definition of retinal microvasculature in OCT-A by motion correction, registration, and averaging of sequentially acquired images will be described. Detailed, high quality OCT-A images are needed for clinical studies such as comparisons of OCT-A with histology and fundus photography fluorescein angiography (FA), and studying the shunting of vessels in a focal area, such as the inner ring of vessels in the foveal avascular zone (FAZ), or in glaucomatous focal defects as shown in this chapter. 16

28 Chapter 3. OCT-A Image Strip-based Registration Non-invasive, in vivo visualization of the retinal microvasculature using OCT-A can be instrumental in studying the onset and development of retinal vascular diseases. For example, OCT-A has enabled the visualization of the deep plexus layer and furthered the understanding of diseases such as paracentral acute middle maculopathy [44] [46] and diabetic retinopathy [47], [48]. Quantitative measurements, such as capillary density, can be used to stratify the risk of disease progression, visual loss, and also for monitoring the course of disease [9], [49]. As mentioned in the previous chapter, due to projection artifact and poor contrast it is often difficult to trace individual vessels in this layer when only one en face image is visualized. An additional challenge to this end is the small dimension and pulsatile flow of the retinal capillaries, making them less consistently visible and difficult to distinguish from the speckle noise relative to larger vessels. This limits the detection sensitivity for changes in the retinal microvascular circulation due to diseases, aging, or treatment. Methods for reliable visualization of the microvasculature in the OCT-A images are required for studies conducting longitudinal and cross-sectional quantitative analysis. Detailed, high quality OCT-A images are needed for clinical studies such as comparisons of OCT-A with histology and fundus photography fluorescein angiography (FA), and studying the shunting of vessels in a focal area, such as the inner ring of vessels in the foveal avascular zone (FAZ), or in glaucomatous focal defects [30]. Serially acquiring and averaging multiple OCT-A images can be an effective solution for confirming the presence or absence of capillaries as the discontinuous appearance of the capillary vessels is beyond improvement simply by just applying image filtering [23], [50]. A crucial step in the serial acquisition approach is the registration of multiple OCT-A images, the difficulty of which is compounded by the fact that an OCT-A image is acquired over multiple seconds and thus particularly susceptible to motion artifacts. The registration of sequential B-scans [51] can aid in attenuating small motion artifacts, but not the larger motion artifacts associated with imaging subjects with pathologies. OCT-A with eye-tracking has been implemented in commercial retinal imaging systems, although this increases hardware cost and 17

29 complexity on which the sensitivity and reliability of motion detection also depend. Previous works on post hoc motion artifact removal by en face summed volume projection (SVP) have been reported in the Literature, see for example [23], [52], [53]. In Hendargo et al. [52], two to three sets of orthogonal (x-fast and y-fast) volumes were acquired and divided into motion-free strips. The visualization and contrast of the vessels were improved by multi-resolution Gabor filtering, and the strips were registered one-by-one, first globally by x- and y- translation that maximized the correlation in the overlapping region, and locally by B-spline free-form deformation in the overlapping region. Zang et al. [53] did not acquire orthogonal data sets, but instead serially acquired two OCT-A volumes in the same scan orientation that were divided into parallel motionfree strips. The strips were first registered by x- and y-translation and rotation that minimized the squared difference of large vessels, which were defined in the paper as pixels with decorrelation value greater than 1.3 times the mean value. This was followed by B-spline free-form deformation on small vessels, defined as pixels with decorrelation value less than 1.3 times and greater than 0.6 times the mean value. Both groups presented mosaicking of OCT-A images into widefield views, which has been reported in other works as well [51], [54]. In this Chapter, averaging of up to 10 serially acquired OCT-A images with parallel strip-wise microsaccadic noise removal and localized nonrigid registration is presented. Unlike the previous two methods [52], [53] which concentrated on motion artifact removal and widefield imaging, our purpose was to improve the contrast and signal to background of the capillaries in focal regions. The details of our methodology are presented below. In brief, the serially acquired OCT-A images were divided into microsaccade-free strips. The target strips were first aligned to a template image by x- and y-translation based on maximum cross-correlation, followed by affine registration using Scale Invariant Feature Transform (SIFT) [55], a feature extraction method robust to scaling, orientation changes, illumination changes, and affine distortions. The image warping and local distortion due to slower eye movements are less obvious and more difficult to model than the strong stripe artifacts from microssacadic motion. Instead of free-form deformation [52], [53], our approach optimized the intensity value at each pixel location as the average of the values from each overlapping strip determined by translation and rotation of a windowed region in each strip. Thus pixel- 18

30 wise correspondence across multiple OCT-A images was found by local neighborhood matching. The remainder of this Chapter is organized as follows. The Methods section describes our processing algorithm for OCT-A image averaging in detail, as well as quantitative metrics to evaluate the improvements to the image quality. The algorithm was tested on OCT-A images of six healthy volunteers, with the vessel visibility improvement qualitatively demonstrated in all, superficial, and deep plexus layers. Quantitatively, the algorithm performance was evaluated by contrast to noise ratio (CNR) and signal to noise ratio (SNR) with the background speckle noise information from the foveal avascular zone (FAZ). The Chapter ends with a discussion of the image quality improvements using our method of averaging serially acquired OCT-A images Methods All subject recruitment and imaging took place at the Eye Care Centre of Vancouver General Hospital. The project protocol was approved by the Research Ethics Boards at the University of British Columbia, Simon Fraser University, and Vancouver General Hospital, and performed in accordance with the tenets of the Declaration of Helsinki. Written informed consent was obtained from all subjects Optical Coherence Tomography Instrumentation The OCT-A images were acquired from a GPU-accelerated OCT clinical prototype, previously described in Section Ten serially acquired volumes centered at the foveal avascular zone (FAZ) were obtained per eye in ~32s. During this image acquisition period, patients were asked to maintain their gaze on a particular target, and encouraged to blink as necessary in order to prevent drying of the cornea. The automated parsing of the image data strips (Section 3.2.1) eliminated issues of motion artifact and partial volumes En face Angiogram Extraction Post-processing of the raw intensity data was performed to extract optimal quality images of the retinal microvasculature according to the procedure outlined in Section 19

31 Projection artifacts in the deep layer angiogram were attenuated using a modified slab-subtraction algorithm [56]. In Equation (2, PR Deep is the projection resolved en face angiogram of the deep layer, where Norm represents the normalization process, N Deep is the number of pixels in the deep layer, N All Layers is the number of pixels in all the retinal layers, N Superficial is the number of pixels in the superficial layer, and sv is the angiogram PRDeep Norm sv * Norm sv Norm sv Deep All Layers Superficial N Deep NAll Layers NSuperficial. (2) 3.2. Angiogram Registration The algorithm overview is shown in Figure 3.1. The ten serially acquired en face images of all retinal layers were divided into microsaccade free strips, which were then registered to a template image, first using rigid registration for the course alignment, followed by non-rigid registration for finer features. Transforms applied to the en face image of the full retinal thickness were then applied to both the superficial and deep layer angiograms. Figure 3.1 Overview of the strip-based registration algorithm for multiple serially acquired OCT-A images. Representative images are used to demonstrate the algorithm in Figure Microsaccade Free Strip Generation For each eye, a microsaccade-free image from the ten en face images was chosen as the template image. In the case that all images contained microsaccadic motion artifacts, a template was generated by stitching together microsaccade-free strips using the registration methods discussed below. 20

32 After the template image was chosen / generated, the remaining images were divided into strips between positions in the image corresponding to where the patient fixation was lost, which appeared as vertical white stripes in the en face image, as shown in Figure 3.2. Strips less than 40 pixels wide often contained large drift artifact and were therefore discarded. If multiple microsaccade free images existed per eye, the first was selected as the template and the rest were divided into three equal-sized strips for registration. Each strip was zero-padded to match the size of the template image and coarsely aligned to the template by x- and y-translation using maximum cross correlation. Figure 3.2 Demonstration of the image stripping, coarse translation, affine registration and non-rigid registration steps of the proposed algorithm. The template image (green) and registered strip (magenta) are shown as composite images where white regions indicate where the two images have the same intensities. The areas under the red, orange and yellow boxes are further explored in Figure

33 Figure 3.3 Comparison of three different strips registered to the same template image using (a) coarse translation, (b) affine registration, and (c) non-rigid registration. The template image (green) and registered strip (magenta) are shown as composite images where white regions indicate where the two images have the same intensities Strip-based Affine Registration Scale Invariant Feature Transform (SIFT) keypoints were automatically extracted from both the template image and each strip to be registered [55]. Briefly, keypoints are the locations of local scale-space extrema in the difference-of-gaussian function convolved with the image. Further refinement to the keypoints can be made by assigning each keypoint an orientation to achieve invariance to image rotation. Finally, a local image descriptor is assigned to each keypoint using the image location, scale and orientation as found above. Readers are encouraged to refer to [55] for a more detailed description of the SIFT algorithm. As the SIFT feature descriptor is invariant to uniform scaling and orientation, it is ideal for identifying matching keypoints in noisy or speckled images such as OCT-A angiograms. The calculation of Euclidean distances in MATLAB is computationally expensive, and therefore matching keypoints between the template and strip were identified as the closest corresponding keypoints by a small angle approximation to the Euclidean distance. Keypoints were considered matching if the ratio of the vector angles from the nearest to the second nearest match was less than a threshold value of

34 As the image had been coarsely aligned in the previous step, a second check was included to ensure the matched keypoints were no more than 40 pixels distant in the x or y direction. All strips that had a minimum of 4 matched keypoints were then transformed using an affine transform estimated using the matching keypoints as inputs to the EstimateGeometricTransform function in MATLAB. This function iteratively compares an affine transformation using three randomly selected keypoints, where the transformation with the smaller distance metric calculated using the M-estimator SAmple Consensus (MSAC) algorithm is used as the transformation matrix for the next comparison. The maximum number of random trials for finding the inliers was set to 5000 for improved robustness Strip-based Non-Rigid Registration The vertical white lines in the target image in Figure 3.2 mark the image discontinuities due to microsaccades accounted for by the strip-based affine registration; however, localized mismatch still remains in the aligned images after this affine registration step. The next step in our algorithm is to compensate for the smoother tremor and drift motions represented by image warping and distortion, by using non-rigid registration. Prior to non-rigid registration, a 2x2 averaging filter was applied to both the template and the aligned strip to smooth any fine speckle that may affect the non-rigid registration. The template and aligned strip were both then zero padded by 15 pixels. For each pixel in the strip the normalized cross-correlation[57] was calculated, defined by, xcorr norm ( s, t) [ f ( x, y) f ][ m( x s, y t) m] xy, st, 2 2 [ f ( x, y) f, st, ] [ (, ) ] x y m x s y t m x, y, (3) where f(x,y) is the 29x29 pixel matrix field centered on the (x,y) pixel of the template image, f s,t is the mean of the image in the region under the mask, m is the 15x15 pixel mask matrix centered on the pixel of the strip and m is the mean of the mask. This was also done for -15,-10,-5, 5, 10, and 15 rotated field matrices. The pixel located at the index of the maximum normalized cross correlation was then used as the registered pixel for the strip. Figure 3.2 shows a pictorial schematic of the registration pipeline 23

35 described in this section. A smaller field of view demonstration of the coarse, affine and non-rigid registration steps is shown in Figure 3.3. The stack of registered strips could then either be combined by taking the mean or median to generate a higher quality image Validation The performance of the algorithm was evaluated with qualitative observation and quantitative measures of the contrast to noise ratio (CNR), signal to noise ratio (SNR), and structural similarity index (SSIM). The CNR [58], [59] is defined as CNR 10log r b, (4) 2 2 r b where r and σ 2 r are the mean and variance of the whole image b and σ 2 b are the mean and variance of the background noise region. The background noise region was selected to be the largest rectangle that would fit within the FAZ. As this is an area of non-perfusion, any signal located here in a healthy eye can be considered noise. Vessel segmentation to delineate the pure signal34 was not used here, as the quality metric was only used for intra-volume comparison to measure the trends. The SNR [58], [59] is defined as max( X ) SNR 10log lin 2 lin 2, (5) where X lin is the matrix of pixel values in the angiogram on a linear intensity scale and σ 2 lin is the noise variance on a linear intensity scale. The background noise region selected was the same used in the CNR calculations. The SSIM [60] is a quality metric used to measure the perceived relative quality of a digital image, and is defined by 24

36 SSIM ( x, y) (2 c )(2 c ) x y 1 xy ( x y c1)( x y c2), (6) where x is the image to be compared, y is the final averaged image, and, σ 2 and σ are the average, variance and covariance respectively. The terms c 1 and c 2 are small constants << 1 added to avoid instability when 2 x + 2 y or σ 2 x + σ 2 y are equal to zero Results A total of 10 eyes from 6 healthy volunteers (4 male, 2 female) aged 36.8 ± 9.3 years were acquired according to the imaging protocol. A comparison of the template image and the final averaged OCT-A images for all retinal layers, as well as the superficial and deep vascular layers is shown in Figure 3.4 and Figure 3.5. In the template images, the vessels near the FAZ are relatively clear; however, it becomes harder to differentiate the vessels further towards the periphery. In contrast, the vessels in the averaged images are clearly seen throughout. Improvement in vessel visibility is particularly marked in the deep layer, where the OCT signal strength is weaker. Qualitatively the median images appear sharper than the mean images as the median averaging acts as a speckle reducing filter. However, the mean images appear more smooth than the corresponding median image. 25

37 Figure 3.4 Template image, mean, and median averaged images (all retinal layers, superficial and deep plexus) for Subject 3 OD, a healthy male subject, 29 years of age. 26

38 Figure 3.5 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. 27

39 Figure 3.6 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a subject with diabetic retinopathy. For quantitative comparisons of the template and final averaged images, the average CNR and SNR of the images was calculated. The average CNR of the angiograms with all retinal layers increased from 0.52 ± 0.22 db using the template images to 0.77 ± 0.25 db with the mean images, and 0.75 ± 0.24 db with the median images. Additionally, the average SNR of the angiograms with all retinal layers increased from ± 4.04 db using the template images to ± 4.73 db with the mean images, and ± 4.89 db with the median images. The mean improvement of both the CNR and SNR was statistically significant (p<0.01) using a paired t-test. To evaluate the change in perceptual quality per strip, the SSIM was calculated on each incremental averaged image of the template and registered strips. Although 10 volumes were acquired per eye, the number of microsaccades and strips less than 40 28

40 pixels in the corresponding en face images was different for all eyes and therefore the number of strips used to generate the averaged images was not necessarily equal. The mean number of strips per eye was 21 ± 7 strips. As seen in Figure 3.7, the SSIM values show a rapid increase as the first few strips are registered and applied to the template image, and then the rate of improvement slows with additional registered strips. This trend was observed in both the mean and median averaged images. Figure 3.7 SSIM values for incremental averaged images for all eyes for the all retinal layers, superficial layers and deep layers Discussion The major findings in this paper are as follows: averaging multiple registered sequentially acquired OCT-A images (1) qualitatively enhances the visualization of the retinal microvasculature networks, (2) increases the SNR and CNR of the angiograms, and (3) increases the perceptual visual quality when using SSIM as a metric. After averaging multiple en face images, the vessels of the deeper capillary plexus are more readily identified, making quantification more reliable and thereby facilitating investigation of its role in the pathophysiology of retinal vascular disease. Although minimal projection artifact can still be seen in Figure 3.4 corresponding to the 29

41 larger superficial vessels, the overall qualitative condition of the en face images is improved. The SNR and CNR both increased significantly by averaging the individual strips. Although both the SNR and CNR of the mean images are larger than those of the median images, there is no significant difference between the mean and median and therefore no recommendation of an averaging method can be made based on these metrics. The SSIM is a full reference metric where the final averaged image was taken to be the perfect quality reference image. As shown in Figure 3.7, the SSIM increases with each additional registered strip that is averaged to the template image. Note that each of the 10 volumetric sets for each subject was divided differently into strips (based on the motion), therefore a different number of strips was used for each averaged reference image. As expected when averaging images, the first few strips applied to the template affected the SSIM the most whereas the later strips provided only modest improvement to the SSIM. The deep plexus showed the greatest increase overall. By increasing the visibility of individual vessels, this technique has the potential to improve automated segmentation results thereby improving our ability to quantify capillary density in normal and diseased states. Although the ability to enhance the visualization of the retinal plexuses through averaging multiple sequentially acquired OCT-A images was demonstrated, there are several limitations of this work which should be acknowledged. This study assessed only relatively young subjects with clear ocular media and good fixation ability. The presence of media opacities in older subjects may limit the amount of capillary information that can be attained from images. Although the algorithm attenuates non-microsaccade motion in the registered strips, the template may contain distortions and image warping which is not accounted for here Summary In this chapter, a pipeline for the registration of sequentially acquired OCT-A images was presented to increase the quality of visualization. This improves one of the 30

42 main limitations in the previous chapter, and in the next chapter we will discuss how the burned of manual segmentation was overcome using machine learning. 31

43 Chapter 4. Automated Quantification Early efforts aimed at OCT-A perifoveal capillary density quantification utilized manual vessel tracing methods [26], but the process can be labour intensive and subject to intra- and inter-observer variability [61]. Fully automated techniques are being explored, but face challenges such as variable intra- and inter-image signal to noise ratios, projection artefacts from outer layer vasculature on to deeper layers, and motion artefacts [31], [62]. OCT-A signal intensity thresholding has been the foundation of most automated segmentation efforts thus far and progress has been made in applying additional filters for more accurate results [49], [63]. Improvements in automated segmentation and quantification of OCT-A images of the retinal vasculature may aid in its wider spread adoption and potential application Our group has demonstrated a novel automated deep learning method to segment and quantify retinal images from a prototype OCT-A machine using Deep Neural Networks (DNN) [31]. The application of the algorithm has been expanded to include OCT-A images from a commercial system, the RTVue XR Avanti (Optovue, Inc) [64]. In this Chapter, the use of the deep learning method for the quantification of Foveal Avascular Zone (FAZ) parameters and perifoveal capillary density of prototype and commercial OCT-A platforms in both healthy and diabetic eyes is evaluated Methods The protocol for this study was approved by the human research ethics committees of Simon Fraser University, the University of British Columbia and the North Shore Long Island Jewish Health System and conducted in compliance with the Declaration of Helsinki. Written informed consent was obtained from all subjects. Patient imaging using the commercial device was performed at Vitreous Retina Macula Consultants of New York from February 13, 2015 to April 25, Patient imaging using the prototype device was performed at the Eye Care Centre in Vancouver, British Columbia from July 14, 2014 to October 13, Data analysis for this study was performed from February 25, 2016, to November 11, The subjects underwent a 32

44 standard ophthalmic examination and their level of retinopathy was determined by the treating physician using the Early Treatment of Diabetic Retinopathy Study (ETDRS) [65] staging Inclusion Criteria Subjects classified as diabetic were diagnosed with diabetic retinopathy according to the ETDRS criteria by an experienced retina specialist. Subjects that comprised the control group showed no evidence of retinal or ocular pathology on examination. All subjects were screened for clear ocular media, ability to fixate and were able to provide informed consent prior to imaging Optical Coherence Tomography Instrumentation Two OCT-A systems were used in this study: one prototype and one commercially available machine. The clinical prototype OCT-A system used in this study was previously described in Section The commercial OCT-A system used in this report was the RTVue XR Avanti (Optovue, Inc) which is an 840 nm spectral domain system with an A-scan rate of 70 khz. The XR Avanti has a reported axial resolution of 5μm and a transverse resolution of 15μm [66] Imaging Protocols Standard imaging procedures differed among the two OCT-A systems used. For OCT-A using the prototype system, the protocol described in Section was used. For the RTVue-XR Avanti system, images were scanned over 3x3mm regions centered on the FAZ with a scan pattern of 2 repeated B-scans at 304 raster positions, with each B scan consisting of 304 A-scans. Two volumetric scans were acquired in this fashion: one horizontally scanned and the other vertically for a total acquisition time of ~6.25s Processing of OCT-A Images The commercial images were processed with the system s built-in image processing software, AngioVue. The split-spectrum amplitude-decorrelation angiography (SSADA) method was used for extracting the OCT-A information. The algorithm split the 33

45 spectrum into 11 sub-spectra and detected blood flow by calculating the signal amplitude-decorrelation between two consecutive B-scans of the same location. Both horizontally acquired and vertically acquired images were registered and averaged Manual Tracing Methods Two trained raters segmented OCT-A images using a Wacom Intuos 4 tablet (SR), a Samsung ATIV Smart PC Pro 700T tablet (FC) and GNU Image Manipulation Program (GIMP). Rater A (FC) segmented all prototype OCT-A images and 26 of the commercial OCT-A images while rater B (SR) segmented the other 18 commercial OCT- A images. The segmentations were reviewed and accepted by two other trained raters (MH and ZM) Algorithm Training Methods The automated segmentation of the blood vessels in the OCT-A images was performed by classifying each pixel into vessel or non-vessel class using deep convolutional neural networks. A detailed description of the DNN architecture has been previously published19. Briefly, original OCT-A en face images and the corresponding manual segmentations were used as inputs to train the deep neural network. An equal number of vessel and non-vessel pixels were extracted from each image to ensure a balanced training set. The trained network then segmented the test datasets by assigning a grayscale value, with higher values representing higher confidence of the pixel being a vessel. The prototype and commercial devices were trained separately due to inherent differences in the original images such as the image size. Both datasets were trained on a mixed training set comprised of both healthy and diabetic images. The first half of the dataset was used to train the deep neural network, which was then used to segment the second half of the dataset. The process was repeated with the datasets reversed. As the commercial training dataset comprised of two separate raters, care was taken to divide the training sets equally between rater segmentations. Due to an icon in the bottom left corner of some data in the commercial dataset, a mask was applied over the area and it was disregarded in further analysis. 34

46 Segmentation Performance Analysis The segmentation performance was evaluated by pixel-wise comparison of the manually segmented images and the thresholded binary output of the deep neural network using Otsu s method for threshold selection. The accuracy, sensitivity, and specificity were calculated for each pixel in the dataset and presented as a mean average. For each dataset the number of true positive (TP), true negative (TN), false positive (FP), and false negative (FN) pixels were used to calculate the accuracy ((TP+TN)/(TP+FP+FN+TN)), sensitivity ((TP/(TP+FN)), and specificity (TN/(TN+FP)) Clinical Outcome Measures Four FAZ morphometric parameters (area, maximum and minimum diameter, and eccentricity) as well as perifoveal capillary density were calculated from the automated segmentation results. The foveal vascular zone was found as the largest connected non-vessel area. The centroid for this area was then used to determine the maximum and minimum diameter. Eccentricity was calculated as e = 1 b2 2 where b is the minimum radius and a is the maximum radius of the ellipse made by the maximum and minimum diameter. Before capillary density measurements were calculated for the automated segmentations a gamma correction filter was applied to ensure vessel connectivity after binarization. Additionally, all erroneously segmented pixels within the FAZ area were set to a non-vessel classification. Perifoveal capillary density was then calculated as the proportion of measured area occupied by pixels which were classified by the algorithm as a vessel. a Paired t-tests and Intraclass Correlation Coefficients (ICC) were used to compare the means and agreement between segmentation methods, respectively, of the four FAZ morphometric parameters and perifoveal capillary density. A Student s t-test assuming heteroscedasticity was used to compare automatically segmented eyes with and without DR. Results for the prototype and commercial OCT-A systems were assessed separately. 35

47 4.2. Results A total of 71 eyes from 42 subjects were imaged as per the study protocol. 12 healthy subjects (21 eyes) and 5 diabetic subjects (7 eyes) were imaged with the prototype OCT-A system, while 18 healthy subjects (31 eyes) and 7 diabetic subjects (12 eyes) were imaged using the commercial RTVue XR Avanti (Optovue Inc., Fremont, CA) system. The mean age of the prototype subjects was 37.3 ± 12.5 years, consisting of 7 females and 10 males. The mean age of the commercial subjects was 37.9 ± 12.4 years, consisting of 9 females and 16 males. Of the diabetic eyes imaged with the prototype system, the number of eyes with non-proliferative diabetic retinopathy (NPDR) =3, Mild NPDR = 1, Severe NPDR = 2 according to the ETDRS Grading scheme, with 3 eyes exhibiting macular edema and 4 which had previous treatment for DR (laser and/or IVI). Similarly, for the commercial system, the number of eyes with NPDR =3, Mild NPDR = 1, Moderate NPDR = 2 according to the ETDRS Grading scheme, with 3 eyes exhibiting macular edema and 7 which had previous treatment for DR (laser and/or IVI) Deep Neural Network Algorithm Performance An example of the automated segmentation output for both the prototype and commercial systems is shown in Figure 4.1 along with the corresponding original image and manual segmentation. For the images acquired with the commercial system, the accuracy (healthy: 0.796, diabetic: 0.831), sensitivity (healthy: 0.763, diabetic: 0.758) and specificity (healthy: 0.869, diabetic: 0.913) of the deep learning algorithm were calculated. These measures were also calculated for the images acquired with the 1060nm prototype system: accuracy (healthy: 0.797, diabetic: 0.833), sensitivity (healthy: 0.806, diabetic: 0.733) and specificity (healthy: 0.790, diabetic: 0.881). 36

48 Figure 4.1 An example image from both the prototype and commercial systems with their corresponding manual and automated segmentations. As some data within the commercial dataset contained an icon in the lower left corner a mask was applied and can be seen in the lower left corner of the automated segmentation result Clinical Outcome Measures Representative healthy and diabetic images from both systems using both segmentation methods for the FAZ perimeter, minimum diameter, and maximum diameter are shown in Figure 4.2. Table 1 shows the results for the clinical outcome measures in both systems. No significant difference existed between the means of the clinical parameters derived from the manual and automated segmentations of images from the OCT-A systems. All statistical measures are reported in Table 1. 37

49 Table 1. Clinical Outcome Measures OCT-A System FAZ Area (mm 2 ) Minimum FAZ Diameter (mm) Maximum FAZ Diameter (mm) FAZ Eccentricity Perifoveal Capillary Density Prototype OCT-A Healthy (n = 21) Manual Automated T-test ICC Diabetic (n = 7) Manual Automated T-test ICC ± ± p = ± ±0.104 p= ± ± p = ± ± p= ± ± p = ± ± p= ± ± p = ± ± p= ± ± p = ± ±0.015 p= Commercial OCT-A Healthy (n = 31) Manual Automated T-test ICC Diabetic (n = 12) Manual Automated T-test ICC ± ± p = ± ± p = ± ± p = ± ± p = ± ± p = ± ± p = ± ± p = ± ± p = ± ± p = ± ± p = Table 1: The mean (± std) of the clinical outcome parameters (FAZ area, minimum diameter, maximum diameter, eccentricity and perifoveal capillary density) are shown for both healthy and diabetic eyes and both OCT-A systems. The p-value from the paired t-test and the Intraclass Correlation Coefficient (ICC) value is also shown to compare manual and automated methods. For both OCT-A systems, eyes with DR had significantly lower perifoveal capillary density (p<0.01), greater maximum diameter (p=0.04), and greater eccentricity (p<0.01) compared to the healthy normals. There was no significant difference in FAZ area or minimum diameter in either system. 38

50 Figure 4.2 FAZ perimeter (yellow), maximum diameter (green) and minimum diameter (red) shown for example healthy and diabetic data from both systems using both manually and automated segmentations. As some data within the commercial dataset contained an icon in the lower left corner a mask was applied and can be seen in the lower left corner of the commercial automated segmentation results (white) and the lower left corner in the manual segmentation of the commercial diabetic image (black) Discussion This study demonstrates the ability of a machine-learning based automated segmentation algorithm to segment the vessels of both healthy and diabetic eyes imaged with prototype and commercial OCT-A devices. The major findings of the study are: 1) Pixel-wise, the accuracy of the automated segmentation was comparable to that of a manual rater in both OCT-A platforms, 2) Deep learning based segmentation can reliably quantify the perifoveal capillary density compared to manual segmentation across the prototype and commercial OCT-A platforms and 3) Deep learning based segmentation has the capacity to reliably quantify the FAZ area, eccentricity, maximum and minimum diameter compared to manual raters in both OCT-A platforms. The fovea centralis is the anatomical area responsible for the highest visual acuity. With the exception of the foveola, the metabolic demands of the fovea centralis are met by a unique arrangement of inner retinal capillaries. These end-artertial capillaries lack anastomoses which make this retinal eccentricity especially vulnerable to ischemic insult by retinal vascular diseases, including diabetic retinopathy. Perifoveal 39

51 capillary ischemia and FAZ enlargement are well-documented observations of macular ischemia in diabetic retinopathy and are correlated to disease severity and progression. While FA remains the current clinical standard for evaluating macular ischemia, its invasive nature and potential adverse events makes it challenging to incorporate in regular screening and frequent follow-up of patients with diabetic retinopathy. OCT-A is an alternative non-invasive, label-free imaging modality that has been favorably compared to histological representation and FA in in the visualization of perifoveal circulation in healthy subjects and patients with DR. Accurate methods of quantification and analysis of OCT-A images are of great research and clinical interest. Optovue s built-in automated vessel segmentation extrapolates skeletonized outputs to show vessel density maps [67], [68]. Potential limitations to this method include underestimating vessel density in areas with thicker vessels and decreased sensitivity to capillary dropout [69]. A more recently published approach by Schottenhamml et al. [69] takes advantage of a vesselness filter to exploit the interconnective nature of the retinal vasculature and generate more detailed vessel segmentations; however, inaccuracies seem to result with vessel shape at vertices. The deep learning based method used in this report takes advantage of the 2-dimensional spatial structure of training images and was able to accurately mimic manual segmentations. Pixel-wise, the accuracy of the automated segmentation outputs compared to manual ranged from 79-84%, which is comparable to a reported inter-rater manual segmentation of ~83% agreement [31]. Perifoveal capillary non-perfusion is defined as the pathological enlargement of inter-vessel distances between perifoveal capillary networks. Perifoveal capillary nonperfusion represents an important biomarker in the pathogenesis of DR. It has been suggested that the early perifoveal capillary non-perfusion could be present in the absence of obvious diabetic retinopathy on clinical examination [70] OCT-A has been shown to be able to delineate the perifoveal capillary networks precisely and consistently in patients with DR [71]. This serves as a motivation to develop an automated tool that can accurately and reliably quantify the perifoveal capillary density. No statistically significant difference in the means of the perifoveal capillary densities when comparing measurements derived from the automated and manual segmentations was found. In comparing diabetic and healthy eyes, the automated outputs from both systems found a significantly lower (p<0.01) perifoveal capillary density in the DR eyes. This suggests 40

52 that perifoveal capillary density calculated using deep learning based segmentation might be a clinically useful tool for evaluating diabetic retinopathy. The FAZ approximately delineates the location of the foveola within the fovea centralis. The absence of retinal vasculature is believed to help optimize the image on central cones. Increased FAZ area has been well-correlated with decreased visual acuity [9] and the severity of capillary nonperfusion [6], [8] in patients with DR. Although there is high inter-individual variability in FAZ metrics for healthy eyes, longitudinal progression of the FAZ morphology may be a useful biomarker for DR [49]. Improved visualization of the FAZ, enabled by OCT-A has allowed researchers to further study this area as it relates to DR [72], retinal vein occlusion [73], and aging [67]. To assess the clinical utility of the automated segmentations in calculating FAZ morphometric parameters, the FAZ area, minimum and maximum diameter, and eccentricity were calculated using both the manual and automated segmentations. No significant difference existed between the means of the morphometric parameters derived from the manual and automated segmentations. For both systems, the diabetic eyes were found to have a greater FAZ maximum diameter (p=0.04), and greater eccentricity (p<0.01) compared to the healthy normals while no no significant difference in FAZ area or minimum diameter was noted. This lack of correlation is likely due to the high inter-individual variability of FAZ metrics and low number of severe NPDR subjects in the study. A non-circular FAZ shape may be a more reliable biomarker as indicated by the greater eccentricity in DR eyes. This study demonstrates the ability of a deep learning based automated segmentation algorithm to reliably segment the perifoveal microvasculature and provide clinically useful FAZ morphological measures. A limitation of this study is a restricted sample size for the diabetic groups. Additionally, as the performance of a deep learning based approach is limited by the quality of the training data, the automated segmentation performance is limited by image quality and manual vessel segmentations. Common OCT-A image quality issues such as low signal-to-noise ratio within the FAZ and motion artefacts, as seen in Figure 4.3, caused erroneous segmentations in some cases. Although the manual segmentations were reviewed by two trained raters in an attempt to mitigate manual segmentation error, the segmentations were reviewed on a holistic level whereas the machine learns on a pixel-by-pixel basis. Another limitation is the need for a database of segmented images for each field of view and each machine. The commercial machine allows the imaging of three different fields of view (3x3, 6x6, 41

53 8x8mm), of which only one (3x3mm) was chosen to analyze in this study. Experimentally, performance variability in the vessel thickness occurred when the training set and the segmented images were from different fields of view, therefore a training set is needed for each field of view. Figure 4.3 Examples of low quality input data and the automated segmentation. Due to the low signal-to-noise ratio within the FAZ, some areas were erroneously segmented. Additionally, a horizontal motion artefact can be seen cutting through the FAZ which was also incorrectly segmented in areas. Systematic screening of people with diabetes has been shown to be a costeffective approach for identifying potential vision loss [74]. OCT-A is a promising new technology that has the potential to help guide earlier management decisions and prognosis. Deep learning automated segmentation of OCT-A may be suitable for both commercial and research purposes for better quantification of the retinal circulation in healthy subjects and in subjects with retinal vascular disease Summary In this chapter, the use of DNNs for automatically analyzing OCT-A images was evaluated. Using DNNs partially eliminates the need for manual segmentations. Although a certain number of initial segmentations would be necessary to train the machine, once the machine is properly trained manual segmentations would not be needed for future studies. The next chapter discusses possible future studies which could build upon the work presented in this report. 42

54 Chapter 5. Future Work While techniques were presented in this report which help to overcome the image quality and automated segmentation needs discussed in Chapter 2, more can be done to advance the clinical utility of OCT and its derivatives. Some possible suggestions are listed below D Registered OCTA Volumes In order to see the temporal variations of the microvasculature, the individual registered volumes can be evaluated separately instead of averaging. In order to achieve efficient blood flow distribution, vascular shunting can occur which allows the blood to bypass the capillaries in certain areas. For example, in response to cold conditions some shunt vessels dilate to cut off blood flow to the extremities thereby preventing heat loss. Conversely, when exercising shunt vessels to the muscles constrict which pushes blood through the capillary networks where it can deliver oxygen to the muscles that require it the most. This autoregulation happens within eyes as well. By registering the networks, we can observe temporal changes within the eye and perhaps study certain retinal vascular diseases from a different angle Registration of Photoreceptor Images The strip-based registration technique described in Chapter 3 is not limited to the registration of retinal blood vessels. In theory, we should also be able to use the pipeline to register other retinal layers, including the photoreceptors. Figure 5.1 shows two representative images acquired with our Adaptive Optics (AO) OCT system, which provided a high lateral resolution enabling visualization of the photoreceptor mosaic. 43

55 Figure 5.1 Two AO-OCT images of photoreceptors acquired from the same patient in a similar area. Scale bar is 50μm. By averaging these images we should be able to see more circular photoreceptors, and in the case of under sampling, we could potentially be able to resolve features we couldn t see with just one volume. The Retinal Pigment Epithelium (RPE) is a layer of the retina which researchers have not yet been able to readily visualize without the averaging of multiple frames [75] Apply the DNN Framework to Averaged Images Using the framework in Chapter 3, capillaries were more readily visualized. This improvement in CNR and SNR would improve manual rater confidence. As manual segmentations are the input to our DNN automated segmentation tool, an improvement in manual segmentations could result in improved and more accurate automated segmentations. Additionally, if given enough information the DNN could highlight other areas of interest in the OCT-A angiograms, like microaneurysms. Microaneurysms are found in diabetic patients and keeping track of the number of microaneurysms that appear in the macula can be useful in tracking disease progression. Another exciting possibility is that DNN could help us to visualize these networks in three dimensions. As OCT-A is acquired in 3D, it is possible to obtain a volumetric view of the vasculature. Instead of segmenting the layers of the retina and extracting 2D images of the networks, we could obtain volumetric manual segmentations of the 44

56 vasculature on which the DNN could train. Manual segmenters could use either the B- scan or C-scan view in order to properly mark the vessels. Delineating the vessels using this technique would also eliminate the need for projection artefact removal as the computer would be able to learn what is and is not artefact through the segmentations. As the retinal plexus layers aren t completely separate, this would allow clinicians and researchers to be able to interact with the data in a new way. 45

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65 Appendix. Further Examples of Averaged OCT-A Images Figure A.1 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. 54

66 Figure A.2 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. Figure A.3 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. 55

67 Figure A.4 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. Figure A.5 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. 56

68 Figure A.6 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. Figure A.7 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. 57

69 Figure A.8 Template image, mean, and median images (all retinal layers, superficial and deep plexus) for Subject 6 OS, a healthy male subject, 56 years of age. 58