UNIVERSITY OF LATVIA

FACULTY OF PHYSICS AND MATHEMATICS

DEPARTMENT OF OPTOMETRY AND VISION SCIENCE

RETINAL IMAGING AT VARIOUS ILLUMINATIONS

MASTER’S THESIS

Author: Daiga Čerāne

Student ID: dc10014

Supervisors: Ph. D. Pauli Fält

M. Sc. Piotr Bartczak

Assistant Proffesor Pēteris Cikmačs

RĪGA, 2015

ABSTRACT

Master’s thesis is written in Latvian and English, contains 48 pages, 33 figures and 6 tables

and 37 references to literature sources.

Purpose: create a spectrally tunable light source for computer-aided diagnosis of diabetic

lesions and other retinal features.

Methods: A digital micromirror device (DMD) based spectrally tunable light source with a

modified fundus camera and monochromatic camera for recording of images. Images taken of

both healthy human subject and diabetes patients with diabetic retinopathy diagnosis.

Conclusions: Spectrally tunable light source with optimal illuminants is compatible with a

fundus camera for retinal imaging to improve contrast of retinal lesions and features.

Keywords: retina, diabetic retinopathy, fundus camera, spectrally tunable light source, DMD.

CONTENT

ACRONYMS AND ABBREVIATIONS ................................................................................ 1

INTRODUCTION .................................................................................................................... 2

1 LITERATURE REVIEW ................................................................................................. 3

1.1 Fundus of the eye and diabetic retinopathy ................................................................ 3

1.2 History of artificial light sources ................................................................................ 11

1.3 Retinal imaging ............................................................................................................ 14

2 EXPERIMENT ................................................................................................................ 22

2.1 Objective and tasks ...................................................................................................... 22

2.2 Methods ........................................................................................................................ 23

2.3 Subjects ......................................................................................................................... 29

2.4 Results and result analysis .......................................................................................... 31

2.5 Discussion ..................................................................................................................... 40

CONCLUSIONS ..................................................................................................................... 41

ACKNOWLEDGMENTS ...................................................................................................... 42

BIBLIOGRAPHY ................................................................................................................... 43

ATTACHMENT: ANALYZED RETINAL IMAGES ........................................................ 46

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ACRONYMS AND ABBREVIATIONS

ILM – internal limiting membrane

NFL – nerve fiber layer

GCL – ganglion cell layer

IPL – inner plexiform layer

INL – inner nuclear layer

OPL – outer plexiform layer

DM – diabetes mellitus

IDDM – insulin dependent diabetes mellitus

DR – diabetic retinopathy

NPDR – non-proliferative diabetic retinopathy

PDR – proliferative diabetic retinopathy

AD – Anno Domini

BC – Before Christ

IR – infrared

NIR – near infrared

RGB – a color system that consists of red, green and blue channels, that allows to reproduce

wider array of colors

LED – light emitting diode

LASER – light amplification by stimulated emission of radiation

OD – right eye (oculus dexter from Latin)

OS – left eye (oculus sinister from Latin)

fps – frames per second

NA – not applicable

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INTRODUCTION

Diabetes is a disorder that in 100% of cases after 20 years show some kind of retinal

lesions. The diabetic retinopathy is leading cause of legal blindness in people aged 20-74.

Therefore constant supervision of an ophthalmologist is required.

Usually color and standard red-free fundus images of diabetes patients are recorded.

However it is found that these images not always showcase the retinal lesions and features at

the highest possible contrast, therefore it is the aim of this study to create an optimized

(spectrally tunable) light source that could aid in computerized diagnostics of such lesions and

features.

The objective of this study was to create a spectrally tunable light source for computer-

aided diagnosis of diabetic lesions and other retinal features.

Tasks:

1. Create a spectrally tunable light source that can be used in setup with fundus camera

optics.

2. Calibrate the spectrally tunable light source for precise retinal imaging.

3. Obtain images of mechanical model of an eye, determine if RGB images can be

recreated.

4. Obtain images of human subjects suffering of diabetic retinopathy and healthy human

subjects. Determine if contrast of optimal illuminations is an improvement over

standard red-free fundus images.

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1 LITERATURE REVIEW

The literature review shows the basics of eye anatomy, and touches the subject of diabetic

retinopathy. It also shows the history of fundus research, as well as the most commonly used

light sources in fundus imaging in past, as well as currently used illuminations (and filters). To

correctly observe historically available illuminations, a brief history of artificial light sources

is also shown.

1.1 Fundus of the eye and diabetic retinopathy

1.1.1 Basic anatomy of human eye

Basic eye anatomy is separated into two parts: anterior segment: cornea, iris, ciliary body

and lens and the posterior segment that consists of vitreous humor, retina, choroid and optic

nerve head. Choroid, ciliary body and iris make up the uveal tract. The outer segment of the

eye is made of collagen – sclera, corneal limbus and cornea. The inner part of eye – retina,

together with Bruch’s membrane and choroid make up the eye fundus. The structure of the eye

is seen in Fig. 1.1.

Fig. 1.1. Anatomy of eye1

1 http://biology-forums.com/gallery/medium_57728_04_02_13_5_43_26.jpeg

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1.1.2 Retinal anatomy

The retina is structure located at the back of the eye analogue to camera film. The neural

retina processes photons – incoming visual stimuli and converts it into electrical signal, that is

transported to cortex to be analyzed. All parts of retina except for fovea, ora serrata and optic

disc are made up of 10 retinal layers. The photoreceptive cells in retina are called rods and

cones and are located in the outer part of the retina. Cones are responsible for color vision and

in case of normal color vision there are three different kinds of cones (long, medium and short

wavelength peak sensitivity), rods are responsible for mesopic (twilight) and scotopic (night)

vision. (Fig. 1.2.). (1) (2)

Fig. 1.2. Layers and levels of retina. Layers correspond to the anatomical structure. Levels are

distinguished in relation to fundus photography. (3)

Fovea is a part of retina that has the highest density of photoreceptors – cones. There are

no rods in the central part of the fovea. The center of the foveola (Fig. 1.3.) called umbo

produces the highest possible visual acuity. It consists of tightly packed cones (their size is

exceptionally small compared to the rest of retinal cones, where cones in the fovea are at the

size of 1.5µm, but in the peripheral parts of retina – 6µm)2, external limiting membrane and

pigment epithelium, it is separated from inner nuclear layer by the horizontal oblique fibers of

Henle and between nuclei feature Müllerian fibers. (1)

2 http://webvision.med.utah.edu/book/part-ii-anatomy-and-physiology-of-the-retina/photoreceptors/

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Fig. 1.3. Structure of retina in the area of foveola (1)

Ora serrata is a part of eye anatomy where vitreous body is fixed to the retina (one being

around the optic disc, and the other being at the ora serrata, that is the extreme periphery of the

eye). At this point the transition of neural part of retina transforms into non-pigmented

epithelium and pars plana of ciliary body, the internal limiting membrane is continued as

basement membrane. The retinal pigmented epithelium and Bruch’s membrane are continued

as pigmented epithelium of ciliary body and its basement membrane. As seen in Fig. 1.4. (1)

Fig. 1.4. Structure of retina in the area of ora serrata (1)

Optic nerve head is the place where the axons of ganglionar cells combine and make up

the optic nerve. As seen in Fig. 1.5., the external limiting membrane combines with retinal

pigment epithelium that is supported by border tissue of Kuhnt. The border tissue continues to

choroidal level, and further the axons fixate the retina to scleral lamina cribrosa. Also, it is the

other fixation place of vitreous body. (1)

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Fig. 1.5. Structure of retina in the area of optic nerve (1)

1.1.3 Diabetes and diabetic retinopathy

Diabetes Mellitus (DM) is a metabolic disorder that is characterized by elevated blood

glucose level. Elevated blood sugar level may be caused by higher intake of glucose, decreased

cell intake of glucose or lowered blood insulin levels. All these factors make an impact to the

glucose homeostasis. (4) There are two types of diabetes – Type I and Type II, both types are

polygenic in nature – they are caused by genetic predisposition combined with environmental

factors. (5)

Diabetes is the leading factor in the USA for causing legal blindness between the ages of

20 and 74. DM patients are five times more likely to become legally blind than people without

DM. (4)

Type I diabetes patients are diagnosed early – usually before reaching the age of 30. Type

I diabetes mellitus is also called insulin dependent diabetes mellitus (IDDM), the acronym was

dropped in 1995 by American Diabetes Association and from then on has been called Type I

DM. In Type I DM, the production of insulin is almost non-existent, which is due to almost

complete loss of beta cells (always more than 80%), therefore patients with Type I DM are

completely dependent on external injections of insulin, to ensure their survival. The onset of

the disorder is usually triggered by an infection such as chickenpox. At the time of infection, a

virus triggers a response from lymphocytes that destroy pancreatic beta cells, however the

process of beta cell destruction can be also idiopathic. These patients (Type I) usually become

both ketotic and acidotic very easily due to imbalance of sugar and insulin intake. (4) (5)

Type II is an adult onset diabetes. Historically, it’s also called NIDDM – non-insulin

dependent diabetes mellitus. The onset of disorder is steady, and often the early stage Type II

DM is relatively symptomless, making it hard to identify. Upon measurements blood insulin

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levels are normal or even elevated. The reason for raised blood sugar levels are reduced

sensitivity of insulin receptors on cell membranes. (5) Pancreatic beta cells may not be lost

completely, the damage might be caused by pancreatic or other endocrine gland diseases. (4)

The malfunction of cell’s insulin receptors is often caused by an unhealthy lifestyle: lack of

physical exercise and obesity. (5) Therefore patients with Type II DM usually have increased

blood glucose level, decrease of insulin production and partial or complete insulin resistance.

At the time of diagnosing, patients are usually obese (opposite of Type I patients, where patients

are thin), resistant to acidosis and ketosis, as well as diabetic coma. (4) (5)

About 20% of patients with DM experience shifts in the refraction of their eyes. When

patients have high glucose serum levels they experience more myopic refraction that disappears

when blood sugar levels normalize. There are occasions during hypoglycemia when patients

experience more hyperopic refraction, therefore patients experience visual acuity problems with

both their near and far sights. (6)

For DM patients, the risk to develop DR depends on duration of the DM, arterial

hypertension, microalbuminuria, renal failure, pregnancy, dyslipidemia and lifestyle (smoking

etc.) (6), and cataract extraction. In order to prevent the development of DR, strict blood serum

glucose control is required. (4) (7)

Diabetic retinopathy for Type I DM usually doesn’t start in the first 3-5 years after

diagnosis. However statistics show that after 20 years of DM 100% of patients with Type 1 DM

have some form of DR. Furthermore, 50% of these it is proliferative retinopathy. (4) After 30

years of insulin therapy 12% of patients are legally blind. (6) For Type II DM patients the onset

of disease is unknown, therefore statistics are not complete. DR sometimes is the first sign of

diabetes. (4) Statistics show that after 15 years of Type II DM 25% of patients have PDR. After

35 years 67% of patients have PDR. (6) (2)

Pathogenesis of DM include elevated blood sugar level that can alter genetic expression,

therefore altering cellular response, there is free radical formation, neovascularization. (4)

One of the first DM expressions is capillary membrane thickening due to elevated glucose

levels. Over time collagen deposits in the basement membrane of capillaries, pericytes are

destroyed and capillary walls bulge out, which may lead to endothelial cell proliferation. These

bulges are microaneurysms, their formation seen in Fig. 1.6. Microaneurysms are often

accompanied by dot hemorrhages. The differential diagnostics are performed by fluorescein

angiography (FA): dot hemorrhages appear black, but microaneurysms are seen as bright spots,

as their structure is connected to the blood stream. In this stage DR is called NPDR – non-

proliferative diabetic retinopathy. (4) (6) (7) (8)

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Fig. 1.6. Capillary arms fusing together to form microaneurysm (8)

Retinal hemorrhages are divided in two groups by their location and appearance. Flame-

shaped hemorrhages are located in the retinal nerve fiber layer and are caused by the

hemorrhaging of larger blood vessels – arterioles, therefore hemorrhages appear flame shaped.

Intraretinal hemorrhages are caused by bleeding from the end of capillaries and therefore have

blot-dot shape. As seen in Fig. 1.7. (6) (8)

Fig. 1.7. Blot hemorrhages (on the left) and flame hemorrhages (on the right) (8)

Hard lipid exudates can appear in the later stages of NPDR. Hard exudates are associated

with elevated blood lipid levels, they are intraretinal lipid deposits that are caused by lipoprotein

leakage from blood vessels due to breakdown of endothelial tight junctions, and they are formed

mainly in outer plexiform layer by lipoprotein combination with lipid-filled macrophages. In

fundus images hard exudates appear as white-yellow deposits with well distinguished borders.

Seen on the right image in Fig. 1.7. (4) (6) (8)

Macular edema (swelling) is also associated with NPDR. This symptom is better known

to be exhibited by Type II patients, for Type I DM patients it is usually associated with PDR.

Macular edema makes retina thicker, and there are several classifications: thickening of

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macular region (thickness around 500 nm), or thickening of retina in the region within one

optical disc diameter from the fovea, or presence of hard exudates, combined with the

thickening of the retina around 500 µm from the fovea. (2) (4) (7)

Other NPDR symptoms are early neovascularization to compensate for nonperfusion.

This is called intraretinal microvascular abnormalities (IRMA). Overtime IRMA can also

include hemorrhages and dilated retinal veins causing beady veins (Fig. 1.8.) If venous beading

is seen in two quadrants in the fundus image, the condition is called severe NPDR. Cotton wool

spots (aka soft exudates or microinfarcts) are also associated with severe NPDR (Fig. 1.8.).

They are caused by capillary nonperfusion areas – retina has edema in those regions, it is

ischemic and it produces angiogenesis factors. (4) (6)

In the cases of mild and moderate NPDR it is found that grid laser photocoagulation

reduces the risk of loss of visual acuity in all cases of macular edema for about 50-60%.

Photocoagulation scars on retina are seen as white/yellow spots, however with time they may

acquire pigmentation. Photocoagulation is also used to treat microaneurysm leakage. (4) (7)

Fig. 1.8. Pre-proliferative DR. Visible – arterial abnormalities, cotton wool spot, IRMA, beaded vein

and capillary closure. (8)

Proliferative diabetic retinopathy (PDR) is caused by severe neovascularization and

formation of fibrous tissue that extends outwards from retina into the vitreous cavity.

Neovascularization is caused by ischemic response from retina – it produces angiogenesis

factors that lead to formation of new blood vessels in ischemic regions in the retina, on the optic

disc and on the iris (iris rubeosis). PDR neovascularization is severe and makes complex, often

looping structures, unlike in NPDR, where the neovascularization is contained to a small region.

PDR neovascularization is often accompanied by fibrotic growth, which can cause vitreous

body detachment, retinal dragging, and tractional retinal detachment. Newly formed blood

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vessels are easily torn and which causes hemorrhaging in the vitreous body (hemophthalmos).

PDR causes severe visual acuity loss and may lead to legal blindness. (2) (4) (6) (7)

In the cases of severe NPDR and PDR scatter photocoagulation is used to prevent-caused

glaucoma. The complications of this procedure include macular edema, visual field loss and

night blindness. (4) (6)

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1.2 History of artificial light sources

The history of artificial light sources dates back as far as 400’000BC when first humans

discovered fire. More recognizable forms of actual light sources were primitive lamps that were

found around 13’000BC with animal and vegetable fat ‘lamp’ predecessors, up until 5000BC

that was the only form of artificial light, until some form of bioluminescence was discovered

(fireflies trapped in cages). The next bigger leap for development of artificial light was with

Greek oil-pottery lamps, that underwent a development from open oil lamps to closed off in a

century (600-500BC) (9)

Candles as light source are not strictly restricted to a particular time frame. Many sources

contradict, as the definition of first real candle varies. Some sources call first oil lamps as

candles. (10) However some mention only the date when candles were available as we currently

know them – 400AD. (9) Chinese first candle records date to 259-210BC. In Jewish society

candles date back to 165BC, when candles were used in religious rituals. Beeswax was used in

candle making from 15th century, before that the most popular substance was tallow. In China

it was whale fat, Japan – tree nuts. (11) Candle is also the first illumination used to observe

retina. (12)

In 1645 the magic lantern was discovered – it is the first projector type device that was

invented. 1783/4 was the first larger step in development of oil lamps when Ami Argand created

Argand lamp – based on oil lamps, but one that had hollow circular wick, and a glass cylinder

surrounding it. It produced more light than ordinary oil lamps, and more than several candles,

however it consumed more fuel. In 1790 another oil lamp variation was created – Betty lamp,

where wick was integrated, so that drips could run back to reservoir. (9)

In 1792 William Murdock used heated coal to obtain gas, and used it as illumination for

personal use. 1801 was the year electric arc was discovered by H. Davy, it proved to be high

improvement over other illumination sources for theater and street lamps. (9)

In 1825/6 first lamp using incandescence was developed – a limelight. It showed

properties of being up to 83 times brighter than conventional gas lamps, however it was not the

most convenient of illumination sources, and therefore electric arc lamps gained more

popularity. Limelight lamps found their place in theaters where a lens placed in front of lamp

created the known spotlight. (9) (13)

1853 – Kerosene lamp, still using the same technology as other oil based lamps, however

this one used paraffin as fuel. 1878 Electric candle - Jablochkoff candle that consisted of carbon

rods side by side and separated by inert material. Was never made popular as incandescent lamp

became more popular during that time. The credit of inventing incandescent lamp goes to

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Edison who patented the incandescent lamp in 1880, as well as Joseph Swan who demonstrated

carbon filament incandescent lamp to public in 1879. In 1885 – Gas mantle by Welsback. (9)

1894 – Discovery of Argon (Ar), nowadays mostly used in incandescent lamps to provide

inert atmosphere. In 1898 – Discovered Neon (Ne) and Xenon (Xe). Following was discovery

of neon colors – neon red – when current flows through neon filled vacuum tube it glows red.

Blue color – Ar + Hg (1910-1933), 1933 marks discovery of various other neon colors. (9)

1901 – High intensity discharge (HID) lamp was first developed. The first HID lamp was

a mercury lamp, after that followed high and low pressure sodium lamps, as well as metal-

halide lamp. These lamps gave off a line spectrum, some were covered with phosphorus, thus

achieving wider spectrum. The same year Mercury vapor lamp was first discovered. (9) In the

1910s Irving Langmuir developed first gas filled lamp that slowed the tungsten evaporation

from filament and gave off 12-20 lm/W. (9) (14) (15)

In 1926 Edgerton started experiments with Xenon, discovered flashlight. These

experiments mark the beginning of Xenon flash. (16) Xenon flash is used in stroboscope,

flashlight for photography, nowadays – fundus imaging. Xenon lamp SPD comparison can be

viewed in Fig. 1.9.

Fig. 1.9. Xenon flash lamp, halogen lamp and Mercury Xenon lamp SPD comparison3

In 1932 LPS – Low pressure sodium lamps were invented, they were one of the most

energy efficient lamps at the time, modern LPS produces 220 lm/W. In 1937 low pressure gas

3 http://www.photonics.com/Article.aspx?AID=44487

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discharge lamps were discovered – fluorescent lamps. In 1955 there was an introduction of

dichroic filters into lamps to provide cooler beam, by removing IR radiation from the emitted

light. (9)

In 1960 the first laser was patented. The development towards laser was found earlier,

however due to suspicion of threat towards safety (developer had some activity in left-wing

political activities), the patent was classified. At first there were only red He-Ne lasers. In the

same year halogen and metal halide lamps was patented. (9) (13) (15)

In 1965 there was invention of first LED. While nowadays they can be found having

many different properties (wavelength, power), at first there were only red LEDs. After that

green and amber LEDs were produced. Blue and white LEDs were discovered in the 1990s.

Due to low power consumption and long lives they have been gaining popularity ever since

they were discovered (soon after development started to replace other light sources). Today

there are LEDs of both visible spectrum, NIR and IR. All can be found used in fundus camera

as illumination. In the past several years there have been use of organic material in development

of LEDs, new material is called OLED (organic LED), and even more recently – AMOLED

(active matrix organic light emitting diode). (9) (13)

In 1966 High Pressure Sodium lamps were developed. In 1969 – HMI (as patented by

Osram) mercury medium lamp (HMI stands for Hg – mercury (H), Metal (M) and Halogen (I)).

1994 – Sulfur lamp that was made out of sulfur, combined with argon gas. This Sulfur lamp is

known to produce very bright plasma. (9)

In recent years mostly all artificial light sources have highly developed and new

inventions are fewer. In fundus imaging it can be seen that mostly still Xenon flash lamp is still

used, although it was one of the first light sources in commercial fundus imaging.

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1.3 Retinal imaging

1.3.1 History of retinal imaging

The history of retinal imaging begun with ability to observe a living human retina with

ophthalmoscopy (1851), before this time any retinal imaging or the fact that the reflected light

through a pupil sometimes appears black or red was not explained. There had been some

research before 1851 with animal eyes (and applications with human eyes) – contribution from

Purkinje (1825) with concave spectacle glasses acting as mirror, that was not recognized until

much later. (12)

Before the actual invention of the ophthalmoscope, there had been attempts at viewing

retina. It could be seen with experiments in 1704, when Mėry noticed retinal blood vessels in

cat eyes, and in 1825 with Purkinje concave spectacle glasses, as well as 1846 Brücke’s

explanation how every eye’s pupil could be illuminated if the viewing axis coincides with

illumination axis (Cumming published the results in 1846). (12)

After introduction of ophthalmoscope by Helmoltz in 1851 there were developments in

ophthalmoscopes in terms of illumination, which were as follows: candle (used by Helmholtz),

oil lamp, Argand gas lamp, and after invention of incandescent light bulb it was used to

illuminate retina. In some cases there have been unorthodox illuminants, such as directed

sunlight. As the ophthalmoscope gained popularity in ophthalmologist society there were

several new designs for ophthalmoscope. (12)

Earliest mention of fundus photography is by Dr. Rosenburg in 1864 using fundus of

unconscious cat. No results were published. It is recorded that Dr. Rosenburg was not successful

in photographing a living human eye. (3) (17)

In 1886 first fundus photographs of human eye in vivo were taken and published by

Jackman and Webster. The viewed area of the fundus was limited (even though mydriatic

agents were applied), details were not easily discernable and the image was distorted by a large

reflection of cornea. Scientists Jackman and Webster also mentioned that the exposure time was

2.5 minutes – a very long time for retinal imaging. Illumination used was albocarbon

(naphthalene) – that is toxic. The Jackman’s and Webster’s published image is seen in Fig. 1.10.

(3) (18)

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Fig. 1.10. First ever image taken of human eye in vivo. With red arrow area of optic disc is shown, as

well as a major blood vessel. (18)

Espacenet is a patent database that holds in it over 90 million registered patents from

1836. However the first inventions of fundus cameras up until 1941 are not recorded there. (19)

In 1887 L. Howe published an article concerning improvement of recorded film of eye fundus.

He suggested another illumination for retinal observation – an Argand gas lamp. The mentioned

article does not show any images, though the author mentions that the designed setup shows an

improvement over Jackson’s and Webster’s published images in 1886. The same year Barre

wrote of his findings: clear image of optic nerve and optic disc. To his mind it was due to short

camera lens focus (3 in = 7.62 cm), that made the image bright and therefore reducing the

exposure time to 6-10 seconds. (17) (3)

Further development in fundus photography include Gerloff’s 1891 low magnification

photographs and Thorner’s 1903 apparatus (uneven illumination). The first truly discernable

photographs were obtained by Dimmer shown in public and published in period 1899-1907.

Dimmer used carbon arc illumination in his camera. The photographs of these scientist

experiments are seen in the Fig. 1.11. (3)

Fig. 1.11. Fundus photographs of Gerloff (on the left), Thorner (in the middle) and Dimmer (on the

right) (3)

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It can be seen that fundus photography developed quickly, and in 1926 Zeiss Company

marketed the first commercially available fundus camera. It had only 10 degree field of view,

but already had a color film. The photography took only ½ of second of exposure. Its design

was based on Gulstrand’s principles and was designed by Nonderson. The color photography

of eye fundus at the time was not very popular, as it was very expensive at the time. At the time

of Zeiss camera use the only available illumination was still carbon arc, it continued to be so

up until 1940’s. (3)

With the invention of flash tube in 1946, there was possibility of another illumination

source for fundus cameras, it was first used in ophthalmology in 1949 by Rizutti and

implemented in fundus cameras in 1953. (3)

At this time the first patented fundus cameras appeared and are found in Espacenet to this

date. First three patents recorded are during years 1941 (John A. Clarke – A fundus camera),

1952 (John H. Mcmillin – A fundus camera with included fixation means) and 1959 (Noyori

Kimiharu – A handheld fundus camera), though none include illumination’s used or the

schematics. After that time fundus cameras started to get patented more often and their

distribution over the years can be seen in Fig. 1.14., the total amount of patents during those

years is 498. (19)

The first fundus camera schematics from Jackman and Webster include image of eye and

the light path to and from the eye. Fig. 1.12. However it does not show the precise setup of the

camera. (18) Therefore first full camera schematics that are available from the patent database

are from 1975 ‘for Eye fundus camera with focus setting device’ by M. Isao and K. Yoshimi.

In Fig. 1.13. the structure can be seen consisting of multiple parts however explanation of the

structure is not found. (19)

Fig. 1.12. Schematics of Jackman & Webster fundus camera light path (18)

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Fig. 1.13. Schematics for fundus camera patented in 1975 by Isao & Yoshimi (19)

Decade 1971-1980 includes first development of zooming features (1980), accuracy of

feature detection (1980), pupil alignment (1976), elimination of undesired light (1978) as well

as a focusing system (1975). (19)

Starting from 1981 there are at least 2 patents a year recorded in the database

corresponding to the search term ‘fundus camera’ (Fig. 1.14.). In the decade 1981-1990 there

are novelty mentions of IR use in cameras, improving the viewing angle, implementation of

automatic and manual focusing, first mentions of non-mydriatic cameras, ammetropia

correction, stereo images, included LED illumination, first mention of implemented barrier

filters, laser beam scanning, tilting mechanisms, creating more uniform illumination and

removal of peripheral aberrations. 1989 is the year scanning laser ophthalmoscopy was first

introduced. The next decade 1991-2000 shows implementation of RGB laser, feature detection

(such as eyelid), eye detection (OD/OS), mention of polarizing plate use, improved precision

and focusing time, first mention of halogen lamp use, improved alignment for peripheral

photography, mentions of digital photography, as well as simplified, lighter, cheaper and higher

quality cameras. (19)

From 2001 until 2015 patents have more mentions of non-mydriatic cameras, as well as

larger percentage of LED and IR, NIR use in cameras, as well as improved alignment,

performance, view of angle, focusing, and higher quality images, that are smaller in size and

cheaper to produce. During those years three dimensional cameras have become more popular.

Novelty during these years includes pattern recognition, focal matching, and correction of

severe ametropia – even severe astigmatism. (19)

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Fig. 1.14. Fundus camera related patents over the years (19)

In recent years adaptors and alternatives have been appearing. Such as fundus

photography with a mobile phone (2014) (20) or adaptor system for digital single lens reflex

(dSLR) camera. This latter system used a white LED for illumination of retina. (21)

1.3.2 Dyes used in fundus angiography

In 1871 Adolf Baeyer described an organic dye – sodium fluorescein, among many other

dyes, and after ten years it was used for the first time in ophthalmology when Ehrlich used it to

examine flow of aqueous humor. This was followed by Burke in 1910 who examined the fundus

after administration of fluorescein in coffee. Lange and Boyd in 1942 described the circulation

studies of fluorescein by injection of 5% sodium fluorescein in a vein and then observing

various tissue under excitation of purple light. Their paper states that under the magnification

of microscope they can see as the dye stain enters through artery, passes into capillaries, stain

the tissue and then returns to vein. First actual fundus angiogram was that of a cat eye, where

Chao and Flocks in 1959 published findings of circulation time of cat’s retina to be 2 seconds.

The used method was the injection of fluorescein dye in the vein and then the observation of

retina with ophthalmoscope through a cobalt blue filter. That was followed by Mclean and

Maumenee in 1960 when they published the use of angiography to obtain a diagnosis. (3) (22)

The actual technique of fluorescein angiography was published and explained by

Novotny and Alvis in 1961. Their results included actual results with human subjects in vivo.

Their techniques are a standard still used to this day. (3) (22)

Indocyanine green (ICG) dye was first found in 1950s, and approved by FDA (Food and

drug administration) for use in angiography in 1956. First documented phase of fundus ICG

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Patent count per year (keyword: 'fundus camera')

19

angiography (ICGA) was described in 1973. (23) In ophthalmology it is primary used for

choroidal circulation documentation, as it operates in NIR region, and excitation light can

permeate deeper tissue. It is also slightly less popular that fluorescein angiography, as it can be

directly observed, without digital means of recording. Nowadays combined FA and ICGA have

become more popular. (23) (24) ICGA is contradicted for patients with renal and liver and/or

renal failure, as well as for patients with iodine and/or shellfish allergy. Both types of

angiographies are not recommended to pregnant females, even though the dyes have not been

proven to be teratogenic. (23)

1.3.3 Filters in fundus photography

Now known barrier filters are used to present several features of the eye fundus, as we

know what the depth of wavelength penetration for retinal layers is. It is known that blue light

is reflected off of nerve fiber layer. In Fig. 1.15. monochromatic image taken with blue filter

shows the nerve fiber path on the retina. Green light is reflected off of the retinal pigment

epithelium. Therefore all structures that are in the path of this light can be shown, as red

structures appear black in this illumination, green filters are used to improve contrast of retinal

blood vessels, shown in Fig. 1.15. with the red-free filter and improved contrast of

microaneurysm (shown with arrow). As the red light passes through retina it can illuminate

structural changes in choroidal level, it is shown in Fig. 1.15. in the third fundus image – arteries

are not well distinguished, however the choroidal nevus is shown clearly. (3)

Fig. 1.15. The depth of wavelength penetration for retinal layers, and their respective monochromatic

images (3)

20

1.3.4 Multispectral fundus imaging

RGB fundus camera carries information from three channels, however the multispectral

imaging of retina allows for more than one wavelength specific channel. This information is

more wavelength specific, therefore more detailed information is available.

In year 2009 there is a patent for Hyperspectral fundus camera based on tunable

wavelength light source and its description includes image recording of multiple wavelengths,

and the ability to analyze retina by morphology and spectral signatures. In 2010 another patent

was published that includes an option of recreating the SPD of a red-free filter without inserting

the filter. (19)

However up until this date there are no commercially available multispectral fundus

cameras. Though the possibilities of creating multispectral fundus imaging have been attempted

on several occasions and have been successful. These articles involve wavelength specific high

power LED illumination combined with interference filters (25), or white LED combined with

Piezo-Actuated Fabry Perot Interferometer (PFPI) (26) or halogen lamp coupled with narrow

band interference filters. (27)

1.3.5 Modern fundus cameras

In modern cameras there are two different kind of setups: for mydriatic and non-mydriatic

pupil. The setup can also be combined – hybrid fundus cameras. The non-mydriatic cameras

have developed highly in the past decade. (19) They are very useful for screening purposes, but

their disadvantages, such as no stereoscopic capability and artifacts from under and over

exposure, doesn’t allow them to completely replace the mydriatic cameras. However one of the

advantages is that the training for use of the camera can be greatly reduced. (28) (29) (30) When

non-mydriatic camera is used with pupillary dilatation, screening results are further improved.

(31) Modern screening capabilities also include mobile phone that is used as a fundus camera,

when used together with a condensing lens (an ophthalmoscopy technique). (20)

Scanning laser ophthalmoscope (SLO) uses various illumination monochromatic lasers

that differ between a brand and properties it is used for. There are SLO’s that use 488 nm or

795nm lasers – for fluorescent angiography and indocyanine green angiography respectively.

(3) There are also SLO’s that have option of blue and IR reflectance, in those cases they are

equipped with multiple lasers (488nm, 514nm, 788nm and 820nm). (32)

All modern cameras that are commercially available are digital cameras. Most of the

cameras have some kind of non-mydriatic photography available, however there are few that

are purely mydriatic cameras. Most of the cameras still use halogen lamp for observation and

Xenon flash lamp for photography, though there are other observation light sources, sometimes

21

in combination of halogen lamp – such as NIR or IR LED light source. Xenon flash lamp is

rarely replaced by other options, such as white LED.45678910

4 http://www.usa.canon.com/cusa/healthcare/products/eyecare/digital_non_mydriatic_retinal_cameras/cr_2#Specifications 5 http://www.zeiss.com/meditec/en_us/products---solutions/ophthalmology-optometry/retina/diagnostics/fundus-imaging/visucam-pro-nm.html#technical-data 6 http://www.centervue.com/product.php?id=637 7 http://www.topconmedical.com/categories/imaging-retinalcameras.htm 8 http://www.kowa-usa.com/Ophthalmic-Diagnostics/Products.php?Product-Line=Retinal-Cameras 9 http://www.csoitalia.it/en/asp/home.asp?prod=1&dbpID=16 10 http://usa.nidek.com/products

22

2 EXPERIMENT

2.1 Objective and tasks

The objective of this study was to create a spectrally tunable light source for computer-

aided diagnosis of diabetic lesions and other retinal features.

Tasks:

5. Create a spectrally tunable light source that can be used in setup with fundus camera

optics.

6. Calibrate the spectrally tunable light source for precise retinal imaging.

7. Obtain images of mechanical model of an eye, determine if RGB images can be

recreated.

8. Obtain images of human subjects suffering of diabetic retinopathy and healthy human

subjects. Determine if contrast of optimal illuminations is an improvement over

standard red-free fundus images.

23

2.2 Methods

2.2.1 Components of spectrally tunable light source

For calibration purposes a spectrometer Hamamatsu PMA-12 Photonic Multichannel

C10027-01 was used. This spectrometer works also as optical detector. The black correction is

done by a shutter closing within the spectrometer, therefore is precise. Wavelength sensitivity

from 200 nm to 950 nm. Exposure time from 19ms to 64s. Can be controlled by manufacturers

provided PMA software.11

Digital micromirror device (DMD) used in the experiment setup is manufactured by

Texas Instruments. Micromirror array consist of 1024 columns and 768 rows of micromirrors.

Each mirror can be controlled to be in an ‘on’ or an ‘off’ position by being rotated on its

torsional hinges by 12 degrees in either direction (Fig. 2.*d). These settings are controlled by a

custom made computer program called DMD/Hamamatsu scan – v2.0. The software controls

both DMD and records reading from used Hamamatsu spectrometer. The interface of the

software is shown in Fig. 2.*c.

DMD is controlled by patterns – monochromatic BMP files in the size of 1024x768 pixels

(px). Each micromirror by this program can be either activated (a black pixel) or deactivated

(white pixel). The program controls allow user to set different exposure times, and multiple

repeats of measurements and recording of real-time SPD measurements to a text file (using

Hamamatsu spetrometer). The input for the program is a folder that has one or more BMP files

in its directory. Using custom software it was possible to load all the selected patterns to the

DMD’s buffer, and if needed, use those again.

Fig. 2.*d DMD mirrors/pixels in on (on the right) and off state (on the left).12

11 http://www.hamamatsu.com/resources/pdf/sys/SDSS0008E_PMA12.pdf 12 Texas instruments DMD 101: Introduction to Digital Micromirror Device (DMD) Technology, Texas Instruments Incorporated, 2013

24

Fig. 2.*c Interface of DMD/Hamamatsu scan – v2.0

The optimal light source specta that were used for DMD pattern generation were created

during previous research by P. Fält. (33) These optimal light sources were designed to improve

the visibility and contrast of diabetic retinopathy lesions and other retinal features, such as

macula and to improve contrast between arteries and veins. The scientific computation software

MATLAB (The Mathworks, Inc., USA) was used to generate patterns for DMD input. For this

thesis, the optimal illumination patterns were used to improve contrast of hard exudates, dot-

blot hemorrhages, as well as macula and artery/vein feature contrast enhancement.

The light source used was Thorlabs HPLS-30-04, manufactured by Thorlabs, Inc., USA.

HPLS-30-04 is a high power light source that has both UV, NIR and visible spectrum

components. The luminous flux of the light source – 2800 lm, and correlated color temperature

6500K. The focal point of light source is 8.36 mm (measured from tip of cone). (34) The

measured SPD of the light source is shown in Fig. 2.*g.

Fig. 2.*g SPD of Thorlabs HPLS-30-04 and a red-free filter simulated by the DMD light source

25

In the setup a Thorlabs GR50-0305 ruled diffraction grating was used. Thorlabs, Inc.

USA. This diffraction grating has 300 lines/mm.

The liquid light guide used to transmit light from the spectrally tunable light source to the

fundus camera was a LLG0338-4 manufactured by Thorlabs, Inc., USA. This liquid light guide

provides over 70% transmission of light in the visible range, and lower transmissions in near

UV and near IR region – from 340 nm to 800 nm. The core diameter 5 mm. Liquid light guide

operates well in long term, if the temperature is kept within range from -5C to +35C.13

2.2.2 Spectrally tunable light source

Schematics of created the spectrally tunable light source are seen in Fig.2.*a. It consists

of a plasma light source that produces bright white light. Light source was chosen so that even

after spectral component removal by the DMD, the intensity of the illumination is still high

enough to illuminate the retina. Following the light source is a slit (optimal width – 600µm).

The slit is followed by a diffraction grating and the spectrally dispersed light is focused on the

digital micromirror array. Using the micromirror array, light with desired spectral power

distribution can be created and guided to a fundus camera by a liquid light guide. As the

particular light source also has UV spectral components, a UV filter was included in the setup,

due to IR spectral components heat sinks and a fan was used near the slit.

Fig. 2.*a Optical schematics of the created spectrally tunable light source

13 http://www.thorlabs.de/thorcat/19800/LLG0338-4-SpecSheet.pdf

26

Fig. 2.*h shows one loaded pattern (a) and measured output (d). If the pattern is loaded

in the DMD (illuminated as Fig. 2.*, then all the micromirrors corresponding to black pixels,

will be turned in the desired direction (towards the liquid light guide), but the micromirrors

corresponding to white pixels will tilt to reflect the light towards a light trap (as shown in Fig.

2.*h (c)).

Fig. 2.*h Pattern loaded in DMD (a), the spectrum as illuminating the DMD (b), the open

micromirrors (c) and measured output of optimal light (d)

When a particular pattern set is loaded in the DMD/Hamamatsu scan – v2.0 program, the

DMD follows through the input settings – all the loaded patterns are displayed with the set

exposure time (minimum exposure time 19ms), and repeated as many times as indicated. The

last displayed pattern stays unchanged until other settings are loaded.

2.2.3 Calibration of spectrally tunable light source

Calibration of the spectrally tunable light source was done to ensure that the output

patterns would correspond with the determined SPD’s of the algorithms. Calibration of the

wavelength was achieved with 5px and 1px columns (one by one columns changed from shorter

wavelengths to longer wavelengths). Optimal exposure time was found beforehand, so as to

achieve maximum illumination and not to get overexposed data). The Hamamatsu sensor

b a

c d

27

located at the end of the liquid light guide registered the spectral output, while the DMD

micromirrors changed.

Calibration of the intensity was achieved by activating the rows (first row activated in the

middle, then adding a row on top, next time – on the bottom, until all mirrors were activated).

Optimal exposure time was found with maximum intensity – when all the micromirrors were

in active position. As with the wavelength calibration, spectral data was collected with

Hamamatsu spectrometer.

Slit width determines output illumination power (the wider the slit, the higher the

illumination power), and calibration results for wavelength bandwidth (the wider the slit, the

broader bands). It was found in calibration (measuring from slit width of 100 µm to 600µm in

steps of 100 µm), that the wavelength bandwidth is acceptable in for slit width of 600 µm, and

considering that the best illumination power (Fig. 2*o) was achieved at these settings, this slit

width would be used.

Fig. 2.*o Slit width calibration for intensity at slit width 200 µm (on the left) and 600 µm (on the

right)

2.2.4 Fundus camera

The fundus camera optics used are of the fundus camera Topcon TRC-50V, manufactured

in 1983, by Tokyo Optical. Fundus camera had the native illumination and photography light

sources removed from optical path, the same with native photography film camera and relay

lens. Modified fundus camera parts were covered to prevent dust and other foreign particle

entrance. Existing barrier and excitation filters were locked in empty position. Fundus camera’s

astigmatism and ametropia corrective lenses were left for additional focusing and ametropia

correction means. Fundus camera optics schematics are shown in Fig. 2.*b.

28

Fig. 2.*b Schematics of the modified fundus camera used in the experiment (pattern of camera taken

from (19))

Primary calibration means and measurements were performed at the University of Eastern

Finland using a mechanical model of a human eye (Carl Zeiss Meditec, Germany). These

measurements were performed with the above mentioned fundus camera system and spectrally

tunable light source and coupling the system with a Hamamatsu ORCA Flash4.0 LT

monochrome camera. Its properties include low noise, large field of view, fast speed (up to 30

fps). The camera was controlled with native Hamamatsu provided program. For image

recording it is possible to adjust exposure time (auto-exposure also possible), offset and

binning. Possibility to control gain was not optional. Output images were saved to previously

determined location on a computer hard drive in TIFF format (format records the settings

individual to the image taken, such as camera name, date of creation, bit depth, binning,

exposure time, offset and size of image.

For measurements with human subjects a Retiga-4000DC monochrome camera was used

instead of the Hamamatsu camera. The camera has a high sensitivity for low-light applications.

The resolution of the camera is 2048x2048 px. Multiple binning options available, as well as

possible gain control. Camera records image in TIFF format, including information about

settings used during image capture (such as – exposure time, binning, gain and bit depth).14

2.2.5 Safety standards

It was found that the maximum illumination irradiance for white light at the plane of

cornea was 20 mW/cm2. ANSI safety standards allow for 12 minutes of continuous retinal

exposure. (35)

14 http://www.qimaging.com/products/datasheets/Retiga4000dc.pdf

29

2.3 Subjects

Written consent of all the participants in this study was acquired prior to the fundus

imaging. Trials followed the ethical tenets of the Declaration of Helsinki.

In the experiment there were two subjects with pre-existing diagnosis of diabetic

retinopathy and a control subject that had no previous retinal condition diagnosed. Both patients

have records at the Tampere University Hospital. The patients’ RGB fundus images can be seen

in Fig. 2.*e (subject 1) and Fig. 2*f (subject 2) respectively.

Fig.*e 1st subject’s fundus images of the right and left eye (taken 16.03.2015. at Tampere University

Hospital)

Fig.*f 2nd subject’s fundus images of the right and left eye (taken 16.03.2015. at Tampere University

Hospital)

30

Both patients eye fundus have dot hemorrhages, microaneurysms and hard exudates. 2nd

patient also has laser photocoagulation scars. DR lesions seen in Fig. 2.*e and Fig. 2.*f.

Control subject having no prior retinal condition had no available RGB images to display.

Red-free images of control subject were acquired during the experiment with the newly created

spectrally tunable light source. Images with red-free filter of both DR patients were also

acquired for comparison.

Prior to the experiment intra ocular pressure of all subjects was recorded with ICARE

tonometer (sterile, measures pressure without anesthetic). The dilation of subject’s pupils was

acquired with Oftan Tropicamide 5mg/ml, Saten Oy, Finland. After the application of

Tropicamide the intra ocular pressure was monitored periodically.

31

2.4 Results and result analysis

Due to involuntary and voluntary eye movements participants were asked to fixate gaze

on an external fixation means. For all subjects images were taken with spectrally tunable light

and with a red-free filter for contrast comparison. Then images were filtered – overexposed, out

of focus and images without red-free area matches were not analyzed. Due to use of older

fundus camera, the obtained images were never completely uniformly illuminated, therefore for

analysis of retinal features a selected area of image was analyzed (Fig. 2.*i).

Fig. 2.*i image on the left has uneven illumination around the edges and a ring of artefact (green

arrow), image on the left has been cut to avoid the artefacts

Result analysis was done using MATLAB R2014a Student Version. All taken fundus

photographs were normalized against exposure time. Normalization was done by dividing each

pixel of an image with the exposure value and then scaling the value from 0 to 1. Output of this

was monochromatic image where black pixel corresponded to value 0 and white pixel

corresponded to value 1.

Contrast calculation was done before any other image enhancements were performed

(brightness not modified), the only modification to these images was normalization. For

contrast calculation the Michelson’s contrast formula was used (36):

𝐶𝑀 =𝐿𝑚𝑎𝑥 − 𝐿𝑚𝑖𝑛𝐿𝑚𝑎𝑧 + 𝐿𝑚𝑖𝑛

Contrast calculation values were obtained by calculating average brightness of area in the

size of 3x3px.

32

2.4.1 Patterns and SPD’s

Pattern of optimal illuminant for artery and vein detection is seen in Fig. 2*h (a), and its

corresponding SPD, in Fig. 2.*h (d). The patterns used for dot hemorrhaging, hard exudates

and macula can be seen in Fig. 2.*p. Their spectral power distributions and the reference spectra

from P. Fält work (33) can be seen in Fig. 2*q. It can be observed that the actual spectra doesn’t

completely match the reference spectra, however it is due to the sharp peaks necessary for the

reference spectra.

Fig. 2.*p Patterns used for loading in DMD to obtain the optimal spectra (from left: dot hemorrhaging,

hard exudates, macula)

Fig. 2*q Reference spectra (red line) and actual spectra (blue line) for (a) arteries and veins, (b) dot

hemorrhaging, (c) hard exudates and (d) macula

b a

c d

33

2.4.2 Results of a mechanical eye model

First measurements in University of Eastern Finland were attempted with a mechanical

model of eye (Zeiss). Measurement results were recorded with Hamamatsu ORCA camera.

Measurement results do not correspond with a human eye, as the RGB image of the mechanical

eye indicates (Fig. 2.*k) – color of retina, macula, optic nerve and blood vessels does not match

a living human eye. RGB image was reconstructed using MATLAB, the illumination for each

channel were created by the spectrally tunable light source with optimal SPD’s to recreate

natural white light source.

Fig. 2*k. Reconstructed RGB image of mechanical eye model used in experiment

Each of the used illuminations (optimal for artery and vein distinction, blot bleeding, hard

exudate and macula) was used to take images of the mechanical eye model. Images were taken

with automatic exposure time of Hamamatsu ORCA camera (manufacturer provided software)

and binning of 1x1, 2x2 and 4x4 for ten times each time. The result can be seen in Table 2.*z

Table 2.*z.

The result of multiple auto-exposure data of camera at different illuminations

Binning 1x1 2x2 4x4

average exposure time, ms

standard error, ms

average exposure time, ms

standard error, ms

average exposure time, ms

standard error, ms

Arteries and veins 1658,69 2,98 409,51 0,57 101,48 0,08 Blot bleeds 2579,81 3,15 629,01 0,96 155,51 0,12 Hard exudates 1862,96 2,04 459,55 0,50 133,86 0,10 Macula 1588,60 1,96 392,96 0,91 97,34 0,11

34

Considering that the images were taken from a consistent target – a mechanical model of

an eye, it can be concluded that the cameras was relatively stable (Table 2.*z). This indicated

that for the optimal retinal illumination to retain good image quality and not over-tax patients,

optimal binning would be either 2x2 or 4x4 for camera Hamamatsu ORCA.

The actual images or mechanical eye model were normalized and are shown in Fig. 2.*j,

showing images of all above described illuminations.

Fig. 2.*j Mechanical eye model images taken with optimal (a) artery/vein, (b) blot bleed, (c) hard

exudate and (d) macula illuminations

All images show a circular artifact – Fig. 2.*i, image taken with a human subject with

Retiga 4000 DC, as well as the reconstruction of RGB (Fig. 2.*k) and the monochromatic

images in Fig. 2.*j – taken with Hamamatsu ORCA camera. This indicates that the artifact is

due to fundus camera optics, not the camera’s used.

2.4.3 Results of human retina

In some cases for artery and vein analysis the same region of arteries and veins could not

be matched to the same region (control subject). It can be seen in Fig. 2.*l, that below the optic

nerve arteries and veins are not discernable as they fall into unevenly illuminated part of retina,

and therefore those could not be analyzed. For artery and vein analysis in case of optimal

illuminants also region bellow optic nerve head was taken.

b a

c d

35

Fig. 2.*l Red-free illuminations of control subjects left eye

Control subjects images taken with optimal illuminants for artery and vein contrast can

be seen in Fig. 2.*m.

Fig. 2.*m Optimal illumination images for artery and vein contrast improvement for control

subject

The contrast values that were calculated for each region with Michelson’s formula (found

above) for the case of control subject for optimal illuminant for artery and vein distinction can

be found in Table 2.*y. It can be concluded that in case of optimal illumination for control

subject artery and vein contrast was vastly improved, if area of interest was evenly illuminated.

Artery and vein contrast comparison was also done for subject 1 and subject 2. The

contrast comparison result can be viewed in Table 2.*x, the images of selected areas can be

viewed in attachment No. 1. For these subject 1, the artery and vein contrast was improved,

however in case of 2nd subject, the improvement was not seen.

36

Table 2.*y

Contrast comparison for red free filter and optimal illuminant in case of arteries and veins for control

subject

Red-free filter contrast Optimal illuminant contrast

1. Above optic disc 11.53% 8.06% Below optic disc NA 32.91%

2. Above optic disc 1.14% 6.68% Below optic disc NA 24.62%

3. Above optic disc 5.97% Below optic disc NA

Table 2.*x

Contrast comparison for red free filter and optimal illuminant in case of arteries and veins for subject 1

and subject 2

Subject 2 Subject 1

Red-free filter contrast

Optimal illuminant contrast

Red-free filter contrast

Optimal illuminant contrast

1. Above disc 19.97% 21.25%

8.07% 22.32% Below disc 38.29% 22.50%

2.

12.33% 3. 8.17%

2nd subjects right eye was observed to determine contrast enhancement in case of dot

hemorrhages. It can be concluded that dot hemorrhage contrast is improved in case of optimal

illuminant. The images can be seen in Fig. 2.*n, the contrast results in Table 2.*w.

Fig. 2.*n 2nd subject’s dot hemorrhaging images in case of red-free (on the left) and optimal (on the

right) illuminations

37

Table 2.*w

Contrast comparison for red free filter and optimal illuminant in case of dot hemorrhages for subject 2

Red-free filter contrast Optimal illuminant contrast Hemorrhaging on the left 15.12% 18.48% Hemorrhaging on the right 15.43% 19.85%

Images of 1st subject’s left eye were analyzed for contrast comparison for hard exudates.

Fig. 2.*r shows a red-free images and an images of hard exudates for optimal illuminant. The

contrast results for this subject shown in Table 2.*v.

Fig. 2.*r 1st subject’s (a-b) red-free image with three and two hard exudates respectively (for analysis)

and (c-d) optimal illuminant images with three hard exudates, each for analysis

Table 2.*v

Contrast comparison for red free filter and optimal illuminant in case of hard exudates for subject 1

Red-free filter contrast Optimal illuminant contrast Hard exudate location Fig. 2.*r(a) Fig. 2.*r(b) Fig. 2.*r(c) Fig. 2.*r(d) top 36.74% NA 35.59% 87.30% middle 35.70% 43.40% 52.97% 47.17% bottom 46.5% 44.73% 50.29% 37.39%

b a

c d

38

Table 2.*v results show that in case of narrow band illuminants focusing has become

more important, as some features are better focused in one image Fig. 2.*r (d), the top exudate

is most focused, and therefore has the highest contrast. In general for red-free illumination

contrast is moderate, but for optimal illuminants it is possible to achieve higher contrast values,

if image is focused properly.

For control subject the contrast of macula was calculated with both red-free illumination

and optimal illuminant. For right eye, the circular artifact didn’t impact the calculations of

contrast, however for left eye, the artifact covered one edge of macular area, and therefore

contrast calculations were obtained by the opposite edge.

In Fig. 2*s the OD/OS image of red-free and optimal illuminants can be seen, the rest of

the images are included in the 1st attachment.

Fig. 2.*s Fig. 2.*r Control subject’s (a-b) OD and (c-d) OS eye images. (a,c) Red-free images and

(b,d) optimal illuminant images for macula enhancement

b a

c d

39

Table 2.*u shows the contrast differences between red-free and optimal illuminants in

control subject. All cases show that the contrast of macula is improved.

Table 2.*u

Contrast comparison for red free filter and optimal illuminant in case of macula enhancement for control

subject (green cells show contrast of images in Fig. 2*s)

Right Eye Left Eye Red-free filter

contrast Optimal illuminant contrast

Red-free filter contrast

Optimal illuminant contrast

1. 20.28% 43.26% 28.19% 30.71% 2. 11.31% 37.62% 16.17% 3. 27.26%

40

2.5 Discussion

The results of this thesis show that narrow-band illuminations for retinal imaging include

detailed spectral information about the fundus of the eye that improve computer aided

diagnostics, however the implementation of such setup is not easy. Light sources have to have

an output of narrow band illumination that is spectrally tunable and short exposure time.

Most of the research on the subject of spectral retinal imaging, arrive at the conclusion

that brighter illumination is required, as the output narrow-band illumination is quite low, if

compared with input broadband illumination. The spectrally tunable light source created in this

study has high power output illumination that is enough to illuminate the retina even at narrower

bands, however the shorter and longer wavelengths (below 400 nm and above 650 nm) have

lower intensity output.

There have been many other different approaches to this field of study, such as narrow

band filters in front of a broad band illumination (different configurations, such as a wheel or

lath (27)). As well, there have been spectral light sources created by combining high powered

multiple wavelength LED illumination with narrow band interference filters, therefore the

spectral images can be recorded, but the light source is not completely spectrally tunable. (25)

It is mentioned that liquid crystal tunable filter (LCTF) is too slow, when multiple

wavelengths are required. (25) Another way to record specific wavelengths is a computer

tomographic imaging spectrometer that records both spatial and spectral information to a single

file. (37)

More recent study of spectral imaging include a LED light source and Piezo actuated

Fabry-Perot tunable light source that showed good results, however authors mention that

brighter light output would be needed for actual retinal imaging. (26)

41

CONCLUSIONS

The spectrally tunable light source has been constructed and the used optimal

illuminations provide improved contrast, therefore improving possibilities of potential means

of computer aided diagnostics of retinal lesions and feature detection.

Tasks:

1. Spectrally tunable light source has been constructed. The light source can be connected

to a modified fundus camera with a liquid light guide.

2. Calibration of light source has been successful. The parameters of setup have been

modified and the SPD’s of achieved illuminants are closely corresponding with target

illuminant SPD’s.

3. Images of mechanical model of the eye have been obtained. The optimal exposure time

and binning for a specific camera are acquired. Using the spectrally tunable light source

illumination for three separate channels (blue, green and red), allowed to create RGB

image of the mechanical model of the eye.

4. Images of a healthy human subject for retinal feature detection and diabetic retinopathy

patient images for retinal lesions have been obtained. In most cases the contrast of

optimal illuminants were an improvement over red-free filter images. In cases where

contrast wasn’t significantly improved, images were not focused on the lesion or feature,

therefore indicating that the focus of narrow-band illumination requires constant auto-

focusing.

42

ACKNOWLEDGMENTS

This study was funded by Academy of Finland, decision no. 259530.

I thank both of my supervisors from University of Eastern Finland Pauli Fält and Piotr

Bartczak for help in writing this thesis, doing the experiments and processing the data. As well

as for inviting me to participate in the ongoing research in their University.

I thank Niko Penttinen at University of Eastern Finland for creating a software for

controlling the spectrally tunable light source.

I thank Pasi Ylitepsa and Elina Hietanen for their assistance with image recording at their

department of Ophthalmology in University of Tampere.

I also thank my supervisor from University of Latvia – Pēteris Cikmačs for support in

writing this thesis.

Also I would like to thank prof. Māris Ozoliņš for help with terminology and translation

of the Thesis.

I thank for support in writing this thesis to my family, especially those who supported me

asking to read and re-read my thesis before it was completed.

43

BIBLIOGRAPHY

1. Schubert, H. D. Structure and Function of the Neural Retina. [book auth.] M. Yanoff and

J. S. Duker. Ophthalmology. 3rd ed. s.l. : Mosby Elsevier, 2009, p. 1528.

2. Ryan, S. J. Retina. 4th ed. s.l. : Mosby Elsevier, 2006. p. 2731.

3. Saine, P. J., Tyler, M. E. Ophthalmic Photography. 2nd ed. s.l. : Butterworth-Heinemann,

2002. p. 398.

4. Muchnick, B. Clinical Medicine in Optometric Practice. 2nd ed. s.l. : Mosby Elsevier,

2008. p. 398.

5. Walls, L. L. What the Practicing Optometrist Should Know About Diabetes. Optometry

Today. 05 02, 2002, pp. 42-45.

6. Laganovska, G., Klindžāne, M., Rasa, I. Tīklenes pārmaiņas diabēta gadījumā. [grāmatas

aut.] G. Laganovska. Acu slimības. Rīga : SIA "Nacionālais apgāds", 2008, lpp. 356.

7. Schlote, T., Rohrbach, J., Grueb, M., Mielke, J. Pocket Atlas of Ophthalmology. s.l. :

Georg Thieme Verlag, 2006. p. 258.

8. Kanski, J. J. Clinical Ophthalmology. 6th ed. s.l. : Butterworth-Heinemann, 2007. p. 931.

9. Williams, B. A History of Light and Lighting. Professional Lighting Resources. [Online]

1990-1999. [Cited: 05 04, 2015.] http://www.mts.net/~william5/history/hol.htm.

10. Baker, N., Brandt, L., Zakrewski, C. Smith College History of Science. [Online] [Cited:

05 2015, 04.] http://www.smith.edu/hsc/museum/ancient_inventions/candles2.html.

11. Tradition, History & Development of Candles. Waxmelters. [Online] [Cited: 05 04,

2015.]

12. A Brief History of the Ophthalmoscope. CR Keeler, The Royal College of

Ophthalmologists. s.l. : Optometry in Practice, 2003, Vol. 4, pp. 137-145.

13. Csele, M. Fundamentals of Light Sources and Lasers. s.l. : John Wiley & Sons, Inc.,

2004. p. 344.

14. Lamp inventors 1880-1940. The National Museum of American History. [Online]

[Cited: 05 05, 2015.] http://americanhistory.si.edu/lighting/bios/langmuir.htm.

15. Smith, F. G., King, T. A., Wilkins, D. Optics and Photonics. 2nd ed. s.l. : John Wiley

& Sons, Ltd., 2007. p. 506.

16. Harold E. Edgerton. Encyclopædia Britannica. [Online] [Cited: 05 05, 2015.]

http://www.britannica.com/EBchecked/topic/179106/Harold-E-Edgerton.

17. Photography of the Interior of the Eye. Howe, L. s.l. : Transactions of the American

Ophtalmological Society, 1887, Vol. 4, pp. 568-571.

18. Jackman, W. T., Webster, J. D. On Photographing the retina of the living eye.

Philadelphia Photographer. 1886, 265, pp. 340-341.

44

19. Espacnet Patent Search. European Patent Office. [Online] [Cited: 05 05, 2015.]

http://worldwide.espacenet.com/advancedSearch?locale=en_EP.

20. Fundus imaging with mobile phone: A review of techniques. Shanmugam, M. P.,

Divyansh, M., Madhukumar, R., Ramanjulu, R. J., Reddy, S., Y., Rodrigues, G. 9, s.l. :

Indian Journal of Ophthalmology, 2014, Vol. 62, pp. 960-962.

21. Reflex-free Digital Fundus Photography Using a Simple and Portable Camera Adaptor

System. A Viable Alternative. Pirie, C. G., Pizzirani, D., Pizzirani, S. 4, s.l. : Journal of

Visual Communications in Medicine, 2011, Vol. 34, pp. 146-155.

22. Landmarks in the Historical Development of Fluorescein Angiography. Saine, P. J. 1,

s.l. : Journal of Ophthalmic Photography, 1993, Vol. 15, pp. 17-22.

23. Yilmaz, T. The Basics of Indocyanine Green Angiography. s.l. : Association of

Technical Personel in Ophthalmology.

24. A Review of Indocyanine Green Fluorescent Imaging in Surgery. Alander, J. T.,

Kaartinen, I., Laakso, A., Pätilä, T., Spillmann, T., Tuchin, V. V., Venermo, M., Välisuo,

P. s.l. : International Journal of Biomedical Imaging, 2012, pp. 1-26.

25. Multispectral imaging of the ocular fundus using light emitting diode illumination.

Everdell, N. L., Styles, I. B., Cacagni, A., Gibson, J., Hebden, J., Claridge, E. s.l. :

Review of Scientific Instruments, 2010, Vol. 81, pp. 093706-1 - 093706-9.

26. Development of Tunable Fabry-Perot Spectral Camera and Light Source for Medical

Applications. Kaarre, M., Kivi, S., Panouillot, P. E., Saari, H., Mäkynen, J., Sorri, I.,

Juuti, M. s.l. : 3rd International Topical Meeting on Optical Sensing and Artificial

Vision, 2013, pp. 231-237.

27. Extending diabetic retinopathy imaging from color to spectra. Fält, P., Hiltunen, J.,

Hauta-Kasari, M., Sorri, I., Kalesnykiene, V., Uusitalo, H. s.l. : Proceedings of SCIA'09 -

16th Scandinavian Conference on Image Analysis, Lecture Note in Computer Science,

2009, Vol. 5575, pp. 149-158.

28. Webb, J. A. Digital fundus camera offers multiple functions. Optometry Times. 2009,

Vol. 1, 8, p. 12.

29. Screening coverage for diabetic retinopathy using a three-field digital non-mydriatic

fundus camera. Soto-Pedre, E., Hernaez-Ortega, M. C. s.l. : Primary Care Diabetes 2,

2008, pp. 141-146.

30. Meszaros, L. Why non-mydriatic cameras will not replace dilated fundus exams.

Optometry Times. June 2012, p. 28.

31. Systematic screening for diabetic retinopathy with a digital fundus camera following

pupillary dilatation in a university diabetes department. Deb-Joardar, N., Germain, N.,

45

Thuret, G., Garcin, A.-F., Manoli, P., Defreyn, A., Gain, P., Estour, B. s.l. : Diabetic

Medicine, 2007, Vol. 24, pp. 303-307.

32. High resolution fundus imaging by confocal scanning laser ophthalmoscopy in the

mouse. Paques, M., Simonutti, M., Roux, M. J., Picaud, S., Levavasseur, E., Bellman, C.,

Sahel, J. s.l. : Vision Research, 2006, Vol. 46, pp. 1336-1345.

33. Spectral imaging of the human retina and computationally determined optimal

illuminants for diabetic retinopathy lesion detection. Fält, P., Hiltunen, J., Hauta-Kasari,

M., Sorri. I., Kalesnykiene, V., Pietilä, J., Uusitalo, H. 3, s.l. : Journal of Imaging Science

and Technology, 2011, Vol. 55, pp. 030509-1 - 030509-10.

34. Thorlabs. [Online] 03 18, 2011. [Cited: 05 15, 2015.]

http://www.thorlabs.de/thorcat/19700/HPLS-30-04-Manual.pdf.

35. Maximum permissible exposures for ocular safety (ANSI 2000), with emphasis on

ophthalmic devices. Delori, F. C., Webb, R. H., Sliney, D. H. s.l. : Journal of the Optical

Society of America, 2007, Vol. 24, pp. 1250-1265.

36. In Search of a Contrast Metric: Matching the Perceived Contrast of Gabor Patches at

Different Phases and Bandwidths. Peli, E. 23, s.l. : Vision Research, 1997, Vol. 37, pp.

3217-3224.

37. Snapshot hyperspectral imaging in ophthalmology. Johnson, W. R., Wilson, D. W.,

Fink, W., Humayun, M., Bearman, G. 1, s.l. : Journal of Biomedical Optics, 2007, Vol.

12, pp. 014036-1 - 014036-7.

38. Quantitative analysis of multi-spectral fundus images. Styles, I. B., Calcagni, A.,

Claridge, E., Orihuela-Espina, F., Gibson, J. M. s.l. : Medical Image Analysis, 2006, Vol.

10, pp. 578-597.

46

ATTACHMENT: ANALYZED RETINAL IMAGES

Artery and vein comparison for red free filter on the left and optimal illuminant on the right

(subject 2, OS):

Artery and vein images for red free filter (subject 1, OS):

Artery and vein image for optimal illuminant (subject 1, OS):

47

Macula enhancement red free (on the left) and optimal illuminant (on the right) images (control

subject, OD):

Macula images for ref-free filter (control subject, OS):

48

Maģistra darbs “Tīklenes attēlu iegūšana ar dažādiem apgaismojumiem” izstrādāts Latvijas

Universitātes Fizikas un matemātikas fakultātē.

Master’s Thesis “Retinal imaging at various illuminations” was written in University of Latvia,

in faculty of Physics and Mathematics.

Ar savu parakstu apliecinu, ka pētījums veikts patstāvīgi, izmantoti tikai tajā norādītie

informācijas avoti un iesniegtā darba elektroniskā kopija atbilst izdrukai.

With my signature I confirm, that research has been done independently, all the used

bibliography has been shown and the submitted electronic copy of research corresponds with

printed version.

Autore/Author: _____________________________ Daiga Čerāne 25.05.2015.

Rekomendēju/nerekomendēju darbu aizstāvēšanai

I recommend/do not recommend thesis for defense

Vadītājs/Supervisor: Ph.D Pauli Fält _____________________________________________

Vadītājs/Supervisor: M.Sc. Piotr Bartczak _________________________________________

Rekomendēju/nerekomendēju darbu aizstāvēšanai

Vadītājs: Docents Pēteris Cikmačs _______________________________________________

Recenzents/Reviewer: Dr. Skaidrīte Purviņa

Darbs iesniegts Optometrijas un redzes zinātnes nodaļā ______________________________

Thesis has been submitted to Department of Optometry and Vision Science

Dekāna pilnvarotā persona/Dean’s authorized person: metodiķe Dzintra Holsta

_______________

Darbs aizstāvēts Valsts pārbaudījuma komisijas sēdē

______________. protokla Nr. ________________

Komisijas sekretārs: docents, Dr.fiz, Pēteris Cikmačs _______________________________