Clinical Optical Coherence Tomography Angiography...
Transcript of Clinical Optical Coherence Tomography Angiography...
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.
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Approval
Name: Morgan Lindsay Heisler
Degree: Master of Applied Science
Title: 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
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Ethics Statement
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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
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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.
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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 ................................................................................................ 1
1.1. Eye Anatomy ........................................................................................................... 2
1.2. Diabetic Retinopathy ............................................................................................... 4
1.3. Glaucoma ................................................................................................................ 5
1.4. Ophthalmic Imaging Modalities ............................................................................... 5
1.4.1. Fluorescein Angiography ................................................................................ 6
1.4.2. Optical Coherence Tomography Angiography ................................................ 6
1.5. Contributions ........................................................................................................... 6
1.6. Outline of Thesis ..................................................................................................... 8
Chapter 2. Research Motivation ................................................................................. 9
2.1. Methods .................................................................................................................. 9
2.1.1. Visual Fields, Disc Photographs, Peripapillary Optical Coherence Tomography .................................................................................................................. 9
2.1.2. Optical Coherence Tomography Angiography Instrumentation .................... 10
2.1.3. Processing of OCT-A Images ....................................................................... 10
2.1.4. Image Acquisition and Quantification ............................................................ 11
2.2. Results .................................................................................................................. 13
2.3. Discussion ............................................................................................................. 15
2.4. Summary ............................................................................................................... 16
Chapter 3. OCT-A Image Strip-based Registration ................................................. 17
3.1. Methods ................................................................................................................ 19
3.1.1. Optical Coherence Tomography Instrumentation ......................................... 19
3.1.2. En face Angiogram Extraction ....................................................................... 19
3.2. Angiogram Registration ......................................................................................... 20
3.2.1. Microsaccade Free Strip Generation ............................................................ 20
3.2.2. Strip-based Affine Registration ..................................................................... 22
3.2.3. Strip-based Non-Rigid Registration ............................................................... 23
3.2.4. Validation ...................................................................................................... 24
3.3. Results .................................................................................................................. 25
3.4. Discussion ............................................................................................................. 29
3.5. Summary ............................................................................................................... 30
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Chapter 4. Automated Quantification ....................................................................... 32
4.1. Methods ................................................................................................................ 32
4.1.1. Inclusion Criteria ........................................................................................... 33
4.1.2. Optical Coherence Tomography Instrumentation ......................................... 33
4.1.3. Imaging Protocols ......................................................................................... 33
4.1.4. Processing of OCT-A Images ....................................................................... 33
4.1.5. Manual Tracing Methods ............................................................................... 34
4.1.6. Algorithm Training Methods .......................................................................... 34
4.1.7. Segmentation Performance Analysis ............................................................ 35
4.1.8. Clinical Outcome Measures .......................................................................... 35
4.2. Results .................................................................................................................. 36
4.2.1. Deep Neural Network Algorithm Performance .............................................. 36
4.2.2. Clinical Outcome Measures .......................................................................... 37
4.3. Discussion ............................................................................................................. 39
4.4. Summary ............................................................................................................... 42
Chapter 5. Future Work ............................................................................................. 43
5.1. 4D Registered OCTA Volumes ............................................................................. 43
5.2. Registration of Photoreceptor Images ................................................................... 43
5.3. Apply the DNN Framework to Averaged Images .................................................. 44
References ..................................................................................................................... 46
Appendix. Further Examples of Averaged OCT-A Images ............................... 54
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List of Tables
Table 1. Clinical Outcome Measures .............................................................................. 38
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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. ......................... 1
Figure 1.2 Eye diagram [credit: National Eye Institute, National Institutes of Health] . 2
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]. ................................................. 3
Figure 1.4 Fundus photograph showing fluorescein imaging of the major arteries and veins in a human left eye. .......................................................................... 4
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). .. 7
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
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
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 3.2. ...................................................................... 20
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
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have the same intensities. The areas under the red, orange and yellow boxes are further explored in Figure 3.3. ................................................. 21
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. .................................................... 22
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
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
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. . 28
Figure 3.7 SSIM values for incremental averaged images for all eyes for the all retinal layers, superficial layers and deep layers. .................................... 29
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. .............................................................. 37
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). ..................................................................................................... 39
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. ....................................................................................................... 42
Figure 5.1 Two AO-OCT images of photoreceptors acquired from the same patient in a similar area. Scale bar is 50μm. ....................................................... 44
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List of Acronyms
CNR
DNN
DR
FA
Contrast to Noise Ratio
Deep Neural Networks
Diabetic Retinopathy
Fluorescein Angiography
FAZ
FWHM
ICC
ILM
INL
IOP
IPL
OCT
OCT-A
ONH
ONL
OPL
RPC
SIFT
SNR
SSADA
SSIM
VF
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
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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
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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.
1.1. 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]
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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
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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.
1.2. 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.
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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.
1.4. 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).
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1.4.1. 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.
1.4.2. 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].
1.5. 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
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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.
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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.
1.6. 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.
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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.
2.1. 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.
2.1.1. 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.
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2.1.2. 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 full-
width 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 ω0 ≈ 7.3 μm (calculated using
Gaussian optics) corresponding to a lateral FWHM of ~8.6 μm. For the angiogram, the
speckle variance calculation [40] was used
2
1 1
1 1( )
N N
jk ijk ijk
i i
sv I IN N
,
(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).
2.1.3. 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. Sub-
pixel 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
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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.
2.1.4. 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 11.46 ± 4.21 mmHg (5-
15: 12), 13.74 ± 3.21 mmHg (10-17: 14) and 16.17 ± 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 non-
blinded fashion. Manual tracing was performed using the GNU Image Manipulation
Program Version 2.8.14. 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
12
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.
13
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).
14
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.
15
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.
16
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.
17
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
18
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 motion-
free 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-
19
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.
3.1. 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.
3.1.1. Optical Coherence Tomography Instrumentation
The OCT-A images were acquired from a GPU-accelerated OCT clinical
prototype, previously described in Section 2.1.2. 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.
3.1.2. 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
20
2.1.3. Projection artifacts in the deep layer angiogram were attenuated using a modified
slab-subtraction algorithm [56]. In Equation (2, 𝑃𝑅𝐷𝑒𝑒𝑝 is the projection resolved en face
angiogram of the deep layer, where Norm⟨… ⟩ represents the normalization process,
𝑁𝐷𝑒𝑒𝑝 is the number of pixels in the deep layer, 𝑁𝐴𝑙𝑙 𝐿𝑎𝑦𝑒𝑟𝑠 is the number of pixels in all
the retinal layers, 𝑁𝑆𝑢𝑝𝑒𝑟𝑓𝑖𝑐𝑖𝑎𝑙 is the number of pixels in the superficial layer, and 𝑠𝑣 is the
angiogram.
Deep Deep All Layers SuperficialDeep All Layers Superficial
1 1 1Norm * Norm NormPR sv sv sv
N N N
. (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 3.2.
3.2.1. 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.
21
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 3.3.
22
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.
3.2.2. 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 0.75.
23
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.
3.2.3. 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,
,,
2 2
,, ,
[ ( , ) ][ ( , ) ]( , )
[ ( , ) ] [ ( , ) ]
s tx y
norm
s tx y x y
f x y f m x s y t mxcorr s t
f x y f m x s y t m
,
(3)
where f(x,y) is the 29x29 pixel matrix field centered on the (x,y) pixel of the template
image, 𝑓𝑠,𝑡̅̅ ̅̅ 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 �̅� 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
24
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.
3.2.4. 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
2 210log r b
r b
CNR
,
(4)
where r and σr2 are the mean and variance of the whole image b and σb
2 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
2
2
max( )10log lin
lin
XSNR
,
(5)
where Xlin is the matrix of pixel values in the angiogram on a linear intensity scale and
σ2lin 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
25
1 2
2 2 2 2
1 2
(2 )(2 )( , )
( )( )
x y xy
x y x y
c cSSIM x y
c c
,
(6)
where x is the image to be compared, y is the final averaged image, and , σ2 and σ are
the average, variance and covariance respectively. The terms c1 and c2 are small
constants << 1 added to avoid instability when 2x + 2
y or σ2x + σ2
y are equal to zero.
3.3. 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.
26
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.
27
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.
28
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 19.58 ± 4.04 dB using the template images to 25.05 ± 4.73 dB with the
mean images, and 25.02 ± 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
29
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.
3.4. 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
30
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.
3.5. 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
31
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.
32
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.
4.1. 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, 2016. Patient imaging
using the prototype device was performed at the Eye Care Centre in Vancouver, British
Columbia from July 14, 2014 to October 13, 2016. Data analysis for this study was
performed from February 25, 2016, to November 11, 2016. The subjects underwent a
33
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.
4.1.1. 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.
4.1.2. 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 2.1.2. 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].
4.1.3. 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 3.1.2 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.
4.1.4. 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
34
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.
4.1.5. 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).
4.1.6. 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.
35
4.1.7. 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)).
4.1.8. 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
𝑎2 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.
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.
36
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).
4.2.1. 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).
37
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.
4.2.2. 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.
38
Table 1. Clinical Outcome Measures
OCT-A System
FAZ Area (mm2)
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
0.300 ± 0.146 0.279 ± 0.113 p = 0.11 0.956 0.415±0.164 0.366±0.104 p=0.23 0.886
0.485 ± 0.174 0.470 ± 0.130 p = 0.58 0.890 0.516 ± 0.161 0.446 ± 0.088 p=0.13 0.830
0.707 ± 0.229 0.707 ± 0.159 p = 0.97 0.869 0.928 ± 0.164 0.895 ± 0.172 p=0.52 0.859
0.721 ± 0.083 0.740 ± 0.092 p = 0.34 0.854 0.819 ± 0.083 0.852 ± 0.052 p=0.30 0.621
0.381 ± 0.043 0.388 ± 0.015 p = 0.54 0.680 0.311±0.047 0.281±0.015 p=0.09 0.634
Commercial OCT-A
Healthy (n = 31) Manual Automated T-test ICC
Diabetic (n = 12) Manual Automated T-test ICC
0.310 ± 0.122 0.314 ± 0.113 p = 0.39 0.988 0.473 ± 0.295 0.460 ± 0.262 p = 0.49 0.988
0.522 ± 0.119 0.525 ± 0.111 p = 0.66 0.984 0.549 ± 0.190 0.490 ± 0.102 p = 0.16 0.777
0.699 ± 0.132 0.715 ± 0.118 p = 0.09 0.961 0.911 ± 0.280 0.926 ± 0.294 p = 0.48 0.985
0.662 ± 0.067 0.675 ± 0.076 p = 0.20 0.828 0.784 ± 0.069 0.818 ± 0.082 p = 0.20 0.572
0.560 ± 0.049 0.560 ± 0.015 p = 0.99 0.484 0.453 ± 0.093 0.472 ± 0.047 p = 0.27 0.839
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.
39
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).
4.3. 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
40
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 non-
perfusion 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
41
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,
42
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 cost-
effective 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.
4.4. 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.
43
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.
5.1. 4D 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.
5.2. 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.
44
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].
5.3. 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
45
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.
46
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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.
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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.
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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.
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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.
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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.