cone signals and activity in myopia and emmetropia · abstract iii participant group, he testimated...

Cone signals and activity in myopia and emmetropia

Nanyu Zhou

School of Optometry and Vision Science, Faculty of Health

Vision Improvement Domain

Institute of Health and Biomedical Innovation

Queensland University of Technology

A thesis submitted in fulfilment of the requirements for the

Degree of Master of Applied Science (Research)

2013

Keywords

I

Keywords Colour vision

Cones

Emmetropia

L/M cone ratios Myopia

Multifocal visual evoked potential (mfVEP)

Retina

Visual evoked potential (VEP)

Abstract

II

Abstract Myopia (short-sightedness) is a common ocular disorder of children and young

adults. Studies primarily using animal models have shown that the retina controls eye

growth and the outer retina is likely to have a key role. One theory is that the

proportion of L (long-wavelength-sensitive) and M (medium-wavelength-sensitive)

cones is related to myopia development; with a high L/M cone ratio predisposing

individuals to myopia. However, not all dichromats (persons with red-green colour

vision deficiency) with extreme L/M cone ratios have high refractive errors. We

predict that the L/M cone ratio will vary in individuals with normal trichromatic

colour vision but not show a systematic difference simply due to refractive error. The

aim of this study was to determine if L/M cone ratios in the central 30° are different

between myopic and emmetropic young, colour normal adults.

Information about L/M cone ratios was determined using the multifocal visual

evoked potential (mfVEP). The mfVEP can be used to measure the response of

visual cortex to different visual stimuli. The visual stimuli were generated and

measurements performed using the Visual Evoked Response Imaging System

(VERIS 5.1). The mfVEP was measured when the L and M cone systems were

separately stimulated using the method of silent substitution. The method of silent

substitution alters the output of three primary lights, each with physically different

spectral distributions to control the excitation of one or more photoreceptor classes

without changing the excitation of the unmodulated photoreceptor classes. The

stimulus was a dartboard array subtending 30° horizontally and 30° vertically on a

calibrated LCD screen. The m-sequence of the stimulus was 215-1. The N1-P1

amplitude ratio of the mfVEP was used to estimate the L/M cone ratio.

Data were collected for 30 young adults (22 to 33 years of age), consisting of 10

emmetropes (+0.3±0.4 D) and 20 myopes (–3.4±1.7 D). The stimulus and analysis

techniques were confirmed using responses of two dichromats. For the entire

Abstract

III

participant group, the estimated central L/M cone ratios ranged from 0.56 to 1.80 in

the central 3°-13° diameter ring and from 0.94 to 1.91 in the more peripheral 13°-30°

diameter ring. Within 3°-13°, the mean L/M cone ratio of the emmetropic group was

1.20±0.33 and the mean was similar, 1.20±0.26, for the myopic group. For the

13°-30° ring, the mean L/M cone ratio of the emmetropic group was 1.48±0.27 and it

was slightly lower in the myopic group, 1.30±0.27. Independent-samples t-test

indicated no significant difference between the L/M cone ratios of the emmetropic

and myopic group for either the central 3°-13° ring (p=0.986) or the more peripheral

13°-30° ring (p=0.108).

The similar distributions of estimated L/M cone ratios in the sample of emmetropes

and myopes indicates that there is likely to be no association between the L/M cone

ratio and refractive error in humans.

Table of Contents

IV

Table of Contents KEYWORDS………………………………………………………………………... I ABSTRACT……………………………………………………………..….….…… II TABLE OF CONTENTS…………………………………………………………. IV LIST OF FIGURES……………………………………………………………….VII LIST OF TABLES…………………………………………………………..……. IX ABBREVIATIONS…………………………………….……………………..……. X DECLARATION………………………………………………………………… XII ACKNOWLEDGEMENTS……………………………………………….…… XIII CHAPTER 1 LITERATURE REVIEW…………………………………………... 1 1.1 INTRODUCTION…………………………………………………………….. 1

1.1.1 Emmetropization………………………………………………….…… 3

1.1.2 Prevalence of myopia………………………………………...….…….. 5

1.2 FACTORS SUGGUSTED TO INFLUENCE EYE GROWTH………………. 6

1.2.1 Hereditary and genetic factors…………………………………...…….. 6

1.2.2 Environmental risk factors for myopia……………………….….…….. 8

1.2.3 Outdoor activity decreases myopia risk……………………….………. 9

1.3 RETINAL CONTROL OF EYE GROWTH………………………………… 11

1.3.1 Role of inner retina……………………………………………..….…. 11

1.3.2 Role of outer retina…………………………………………….…..…. 12

1.3.3 Role of RPE…………………………………………………...……… 13

1.3.4 Role of Choroid………………………………………………………. 13

1.3.5 Neurotransmitters in ocular growth……………………………..……. 14

1.3.6 Role of peripheral retina……………………………………………… 14

1.4 PHOTORECEPTORS AND MYOPIA………………………………...……. 15

1.4.1 Distributions of the Cones and rods………………………..………… 15

1.4.2 Cone disease and refractive error……………………….…………..… 15

1.4.3 Cone involvement in eye growth………………………..…………… 16

1.4.4 Cone ratios in refractive error………………………………………… 17

1.5 METHODS TO ESTIMATE L/M CONE RATIOS…………………....……. 22

Table of Contents

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1.6 MULTIFOCAL VISUAL EVOKED POTENTIAL…………………………. 23

1.7 COLOUR VISION AND MYOPIA…………………………………………. 24

1.7.1 Colour vision…………………………………………………………. 24

1.7.2 Colour deficiency and myopia………………………………………... 27

1.8 AIMS………………………………………………………………….……... 28

CHAPTER2 EXPERIMENTTAL METHODS…………………………………. 30 2.1 INTRODUCTION…………………………………………………………… 30 2.2 MULTIFOCAL VISUAL EVOKED POTENTIAL FOR L/M CONE RATIO

STUDY……………………………………………………………………… 30

2.3 CALIBRATION……………………………………………………………… 34

2.3.1 Spectral and luminance calibration of CRT and LCD monitor………. 35

2.3.2 Gamma correction of CRT monitor…………………………………... 39

2.4 CALCULATION OF L AND M CONE ISOLATION………………………. 44

2.4.1 Silent substitution…………………………………………………….. 44

2.4.2 The L cone modulation………………………………………...….….. 48

2.4.3 The M cone modulation………………………………………………. 50

2.5 STATISTICAL ANALYSIS………………………………………………….. 51

2.6 POWER ANANLYSIS………………………………………………………. 51

CHAPTER3 CONE RATIOS IN MYOPIA AND EMMETROPIA………...…. 52 3.1 INTRODUCTION…………………………………………………………… 52

3.2 PARTICIPANTS……………………………………………………………... 54

3.3 PROCEDURE…………………………………………………………..…… 55

3.3.1 The mfVEP visual stimuli…………………………………………….. 55

3.3.2 The mfVEP recording………………………………………………… 58

3.3.3 Dichromat pilot testing……………………………………………….. 60

3.3.4 Trichromat experiments………………………………………………. 62 3.3.5 Exporting mfVEP data………………………………………………... 62

3.4 RESULTS……………………………………………………………………. 64

3.4.1 The mfVEP responses of dichromats…………………………………. 64

3.4.2 The mfVEP responses of all the trichromats…………………………. 71

3.4.3 The L/M cone ratios of trichromats…………………………………... 76

3.4.4 Data analysis between emmtropic, low myopic and high myopic groups………………………………………………………………… 77

3.4.5 Data analysis between emmtropic and myopic groups……….…….… 77

Table of Contents

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3.4.6 Data analysis between central and peripheral retina…………..……… 78

3.4.7 Post hoc effect size analysis……………………….…………..……… 78 CHAPTER 4 DISCUSSION……………………………………………………… 79

4.1 CONE RATIOS IN TRICHROMATES……………………………………… 79

4.2 MYOPES VS EMMETROPES……………………………………………… 80

4.3 CENTRAL RETINA VS PERIPHERAL RETINA……………………..…… 82

4.4 VALIDITY OF THE CONE ISOLATING STIMULI……………………….. 82

4.5 LIMITATION OF THE MFVEP STUDY OF L/M CONE RATIOS……..…. 83

4.6 FUTURE DIRECTIONS…………………………………………….………. 85

BIBLIOGRAPHY…………………………………………………….…………… 86 APPENDIX………………………………………………………………………… 96

List of Figures

VII

List of Figures

FIGURE 1.1 The distribution of refractive error in the newborn baby……………. 4

FIGURE 1.2 Red flowers, green leaves and blue sky with and without colour…... 27

FIGURE 2.1 The mfVEP equipment and stimuli used in this study……………… 31

FIGURE 2.2 A single pattern-reversal VEP response………………………..…… 33

FIGURE 2.3 The L/M cone ratios were calculated from the N1-P1 amplitude….. 34

FIGURE 2.4 The spectral distribution of red, green and blue from the CRT and the

LCD monitors…………………………………...………………….. 36

FIGURE 2.5 The luminance output of CRT and LCD monitors driven by the

VERIS system……………………………………………………… 38

FIGURE 2.6 The modelled gamma functions of our CRT monitor………………. 41

FIGURE 2.7 Gamma function and its inverse function for the green gun….……. 42

FIGURE 2.8 Input-output relationship of the red, green and blue guns after the

gamma correction had been finished……………………………….. 44

FIGURE 2.9 An example of the principle of the silent substitution technique…... 45

FIGURE 2.10 The Smith and Pokony 10° fundamentals…………………………... 46

FIGURE 3.1 The standard stimulus of the mfVEP……………………………….. 56

FIGURE 3.2 The stimuli of the L cone modulation………………………………. 57

FIGURE 3.3 The stimuli of the M cone modulation……………………………… 58

FIGURE 3.4 The placement of the electrodes in this study………………………. 59

FIGURE 3.5 The participant sits 37 cm in front of the monitor with natural pupil

and the left eye is occluded……………………………………..….. 60

FIGURE 3.6 The 16 groups exported from mfVEP………………………………. 63

FIGURE 3.7 The summed and averaged mfVEP signals from one protanope…… 64

FIGURE 3.8 The summed and averaged mfVEP signals from one protanope…… 65

FIGURE 3.9 The amplitude of adjusted modulations from on protanope……...… 66

FIGURE 3.10 The summed and averaged mfVEP signals from one deuteranope… .67

FIGURE 3.11 The summed and averaged mfVEP signals from one deuteranope… 68

FIGURE 3.12 The amplitude of adjusted modulations from one deuteranope……. 69

List of Figures

VIII

FIGURE 3.13 The summed and averaged mfVEP signals in the central 3°-13° retina

of all trichromat participants……………………………………….. 73

FIGURE 3.14 The summed and averaged mfVEP signals in the central 13°-30° retina

of all trichromat participants ………….............................................. 75

List of Tables

IX

List of Tables TABLE 1.1 Prevalence of myopia by country……………………………………… 6

TABLE 1.2 The characteristics of some cone diseases………………………......... 16

TABLE 1.3 Cone ratios amongst people with normal colour vision……………… 20

TABLE 1.4 The refraction of participants in three L/M cone ratio studies……….. 22

TABLE 2.1 The specific stimulator parameters of the mfVEP…………………… 32

TABLE 2.2 The RGB values of the L cone modulation…………………………... 49

TABLE 2.3 The RGB values of the M cone modulation………………………….. 50

TABLE 3.1 The colour combinations of dichromat experiments……………......... 61

TABLE 3.2 The corresponding bit number of each modulation………………….. 62

TABLE 3.3 The L/M ratios of all trichromat participants………………………… 76

Abbreviations

X

Abbreviations L Long-wavelength-sensitive

M Middle-wavelength-sensitive

S Short-wavelength-sensitive

R Red G Green

B Blue

D Dioptres ERG Electroretinogram

mfERG Multifocal Electroretinogram

VEP Visual Evoked Potential

mfVEP Multifocal Visual Evoked Potential

OP Oscillatory Potential RPE Retinal Pigment Epithelium

FDM Form Deprivation Myopia LIM Lens-induced Myopia

LIH Lens-induced Hyperopia

UV Ultraviolet

UVA Ultraviolet A

L-NAME NG-nitro-L-arginine Methyl Ester

VIP Vasoactive Intestinal Peptide

ZENK Zif268 Egr-1 NGFI-A Krox-24

mRNA Messenger Ribonucleic Acid

LCA Longitudinal Chromatic Aberration

MC Magnocellular

PC Parvocellular

KC Koniocellular

LGN Laterale Geniculate Nucleus

ISCEV International Society for Clinical Electrophysiology of Vision

LCD Liquid Crystal Display

Abbreviations

XI

CRT Cathode Ray Tube

VERIS Visual Evoked Response Imaging System

ANOVA Analysis of Variance

Declaration

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Declaration The work contained in this thesis represents my own work. To the best of my

knowledge and belief, the thesis contains no material previously published or written

by another person except where due reference is made.

Signature: Nanyu Zhou

Date : 2 July 2013

Acknowledgements

XIII

Acknowledgements I would like to thank my supervisors, A/Prof Katrina Schmid, Prof David Atchison,

Dr Andrew J. Zele and Prof Brian Brown. Thank-you for suggesting this interesting

field of study, no matter what troubles I have, research questions or personal

problems, you were always very helpful to me. Thank you for your patience,

kindness and support in my research.

Thank you to A/Prof Peter Hendicott for the scholarship provided to me. This meant

that I could focus all my efforts on my study, with one heart and one mind, without

worry.

To Pavan Kumar and Marwan Suheimat, my lovely colleagues, thank you for being a

participant in my pilot testing so many times. I appreciate all the time you spent

assisting me. You are fantastic persons and I wish you the best for your research. I

would also like to thank all my participants who generously gave their time to be

involved in my experiments.

Last but certainly not least, thank you to my parents. The research journey can be

very frustrating. Every time I wanted to give up, your concern and support

encouraged me to persevere and succeed. Thank you for supporting my studies

overseas both emotionally and financially.

Literature Review

1

Chapter 1

Literature Review

1.1 Introduction Myopia is the most common refractive error in children and young adults (Lam et al.,

2004; Lin et al., 1999; Vitale et al., 2008); it occurs because the axial length of the

eye is too long for its optical power (Tong et al., 2002). Key questions concern why

myopia develops and the functional significance of an elongated eye. Both genetic

and environmental factors are determinants of myopia (Hammond et al., 2004; Rose

et al., 2008; Saw et al., 2002; Stambolian et al., 2004; Ting et al., 2004; Wensor et al.,

1999). Twin studies and studies of parental myopia indicate a genetic influence

(Hammond et al., 2004). Spending less time on nearwork and more time on outdoor

activities reduces the prevalence of myopia amongst children, suggesting a lifestyle

impact (Rose et al., 2008).

How the eye uses visual inputs from the external environment to determine which

way to grow remains unclear, although it is well known that the retina plays a crucial

role in eye growth (Wildsoet & Wallman, 1995). In particular, the signals and activity

of the cones across the retina are speculated to contribute greatly in the development

of myopia (Chen et al., 2006; Kawabata & Adachi-Usami, 1997; Liang et al., 1995;

Rucker & Kruger, 2006).

A claim has been made that eyeglasses designed to equalize the activities of L and M

cones can reduce the myopia progression rate to a tenth of that observed with using

normal eyeglasses (Neitz & Neitz, 2011). This result implies that the L and M cone

activities may be different between myopia and emmetropia. However, not all

Literature Review

2

individuals with red-green colour vision deficiency with extreme L/M cone ratios

have high refractive errors (Qian et al.,2009). The prevalence of myopia was found

to be less in persons with colour vision deficiencies than in colour normal

trichromats (46% versus 66%); how many of the persons with colour vision

deficiency had dichromatic and anomalous trichromatic defect was not determined.

As dichromats are missing either L cones (protanopes) or M cones (deuteronopes), it

would be impossible to use relative signals of the two cone types to guide

emmetropization. However the case is more complicated for anomalous trichromats

for which one receptor has a peak sensitivity intermediate between those of the

normal receptors (DeMarco et al., 1992).

The L/M cone ratios have been estimated using various techniques, with all requiring

assumptions such as choice of cone fundamentals, corrections for pre-receptoral

filtering, and method for determining isoluminant points (Hood et al., 2002). The

L/M cone ratios in individuals with normal colour vision vary considerably, ranging

from 0.4 to 13 (Albrecht et al., 2002; Bowmaker & Dartnall, 1980; Brainard et al.,

2000; Carroll et al., 2000; Carroll et al., 2002; Cicerone & Nerger, 1989; Dartnall et

al., 1983; Hagstrom et al., 1998; Hagstrom et al., 2000; Hood et al., 2002; Kremers et

al., 1999; Langrová et al., 2007; Nerger & Cicerone, 1992; Otake & Cicerone, 2000;

Pokorny & Smith, 1987; Roorda & Williams, 1988; Usui et al., 1998). There is little

knowledge of whether L/M cone ratios vary with refractive error. Only three papers

that report L/M cone ratios mention the refractive status of the participants (Otake &

Cicerone, 2000; Cicerone & Nerger, 1989; Nerger & Cicerone, 1992). These three

studies found similar L/M cone ratios between myopic and emmetropic participants,

but there were few participants (n ≤ 6).

The present study investigated the L/M cone ratios in the central and peripheral

retina of myopic and emmetropic normal trichromats using the multifocal visual

evoked potential (mfVEP). There are two alternate predictions for the outcomes. One

is that the L/M ratio may be a determinant of myopia progression, and that the L/M

ratio will be greater in myopic individuals (Rucker & Kruger, 2006). The second is

that given the large range of cone ratios reported amongst individuals and the fact

Literature Review

3

that individuals with red-green colour vision deficiency, in particular

deuteranomalous defects, are not all myopic (Qian et al., 2009), there will not be an

association between the cone ratio and refraction. The mfVEP has been used

previously as a method to estimate the L/M cone ratio, it is non-invasive and widely

accepted; the impact of refractive error has not been previously investigated (Hood et

al., 2002).

This review will consider the role of the retina in eye growth control and in particular

the input of the retinal cones to the emmetropization process. A theory concerning

the ratio of the L and M cones and relative activity of cone types both centrally and

across the paracentral retina in myopia development will be discussed. In addition,

what is known regarding the impact of an elongated eye on the cones inputs to the

post-receptoral neurons will be described. The mfVEP was used in the described

research and thus a section on the visual evoked potential is also included.

1.1.1 Emmetropization

Infant eyes tend to exhibit large refractive errors that can be either myopic or

hyperopic and these refractive errors reduce after birth via a process called

emmetropization (Wildsoet, 1997). Figure 1.1 shows the distribution of refractive

error in the newborn baby from three studies (Siegwart Jr & Norton, 2011); there is

large variability in the refractions. Infant animals show similar refraction data, for

instance, new born macaque monkeys and marmosets have hyperopic eyes (Smith,

1998), while baby ostriches and falcons are myopic (Graham & Judge, 1999), and

baby chicks have a broad spread of refractive errors including both myopia and

hyperopia (Varughese et al., 2005; Wallman et al., 1981).

Literature Review

4

FIGURE 1.1 The distribution of refractive error in the newborn baby (Siegwart Jr & Norton,

2011) from data of Cook and Glasscock (1951) (dashed line), Goldschmidt (1969) (solid line),

and Zonis and Miller (1974) (dotted line). The three studies showed a similar distribution of the

refraction in newborn babies: although there is large variability in the refractions of newborn

babies, the typical refraction is slightly hyperopic.

During the postnatal development of the eye, vision is used by the emmetropization

system to minimise the existing refractive error by regulating the length of the eye to

the optics (Norton, 1999; Smith et al., 1994; Wildsoet, 1997). In the hyperopic

refractive state, the focal plane lies behind the retina. The hyperopic defocus

increases the rate of axial elongation of the eye by presumed changes to retinal

biochemistry and then biomechanical changes of the sclera. By contrast, in the

myopic refractive state, the focal plane lies in front of the retina; the myopic defocus

slows the axial elongation rate such that continued growth of the anterior segment

allows the focal plane to move towards the retina (Troilo & Wallman, 1991; Wildsoet

& Wallman, 1995).

Literature Review

5

The emmetropization process is under retinal control (Norton et al., 1994; Troilo et

al., 1987) and uses visual feedback (Rabin et al., 1981). During the postnatal period,

the eye uses visual signals to elongate rapidly; the axial length of the eye typically

matches the focal plane at about 9 months of age. In cases of visual disturbance, for

example if corneal opacification (Meyer et al., 1999) and/or congenital cataracts

(Rabin et al., 1981) are present, emmetropization is disrupted and the eye grows

more rapidly than normal and a form of deprivation induced myopia develops.

Experimentally induced deprivation myopia has been used in animal studies

(Sherman et al., 1977; Wiesel & Raviola, 1977) to investigate eye growth processes.

These phenomena suggest high quality visual signals are required by the retina for

the determination of the required adjustments to the eye’s rate of growth.

1.1.2 Prevalence of myopia

Many studies seeking to determine the prevalence of myopia have been conducted

over the past century (Wilson & Woo, 1989). Usually East Asian countries have high

myopia prevalence, for example, China (89.5% in school children) (Zhao et al., 2000)

and Singapore (79.3% in young adult males) (Wu et al., 2001). Myopia tends to

occur in childhood (early onset myopia) or teenage years (late onset myopia) and

progresses (Fan et al., 2004; Lin et al., 1999), e.g. worsens, for many years. In regard

to gender, both females and males are susceptible to myopia, but some studies report

higher levels of myopia in females (Vitale et al., 2008; Wong et al., 2000). The

environmental conditions seem to have an impact on myopia, because people who

live in rural areas have a lower chance of being myopic than people who live in cities

(Xu et al., 2005). In addition, workers performing occupations with intensive near

work demands have high myopia risk, including microscopists (Ting et al., 2004) and

textile workers (Simensen & Thorud, 2009). Table 1.1 shows the prevalence of

myopia reported in several studies for comparison purposes.

Literature Review

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TABLE 1.1 Prevalence of myopia by country in different age groups.

Country Age Myopia Prevalence Citation

China 5-15 years 89.5% Zhao et al., 2000

Hong Kong 13-15 years 85% Lam et al., 2004

Taiwan 16-18 years 84% Lin et al., 1999

Singapore 16-25 years 79.3% Wu et al., 2001

Malaysia 15 years 34% Goh et al., 2005

USA ≥20 years 33.1% Vitale et al., 2008

Greek 15-18 years 36.8% Mavracanas et al., 2001

Norway 20-25 years 35% Midelfart et al., 2002

Northern Ireland 12-13 years 17.7% O’Donoghue et al., 2010

Mexico 12-13 years 44% Villarreal et al., 2003

Australia 11-15 years 11.9% Ip et al., 2007

England 12-13 years 29.4% Logan et al., 2011

Sweden 12-13 years 49.7% Villarreal et al., 2000

Spain 30-35 years 30.1% Montes-Mico et al., 2000

Rural Mongolia 7-17 years 5.8% Morgan et al., 2006

India 5-15 years 7.4% Murthy et al., 2002

South Africa 5-15 years 4% Naidoo et al., 2003

Chile 5-15 years 7.3% Maul et al., 2000

1.2 Factors influencing eye growth

It is known that both hereditary and environmental factors influence refractive

development (Ip et al., 2008; Lam et al., 2008), however, there is still intense dispute

about which of these plays the more critical role. For any given individual these

factors will have varying relative importance.

1.2.1 Hereditary and genetic factors

A higher prevalence of myopia in Asian countries than in Western countries indicates

Literature Review

7

a racial impact on myopia. In Asian countries such as China and Hong Kong myopes

make up more than 80% of the whole population (Lam et al., 2004; Zhao et al.,

2000). In Western countries such as USA and Sweden the prevalence of myopia is

lower (30-50% of the teenager and adult population) (Villarreal et al., 2001; Vitale et

al., 2008). The varying different myopia prevalence in Asian versus Western

countries suggests Asian people may have genetic predisposition to myopia.

However, it is also possible that some of the difference is due to different lifestyles

and diets (Edwards et al., 1996; Rose et al., 2008).

Myopia development risk varies with family history; having two myopic parents

increases the risk of developing myopia compared to having no myopic patents. A

survey of 7560 Chinese school students aged between 5 to 16 years highlights the

influence of myopic parents on the refractive errors of their offspring (Lam et al.,

2008). Children with two myopic parents were on average 0.95 D more myopic and

children with one myopic parent were on average 0.40 D more myopic than children

with no myopic parents. Also, the progression of myopia was fastest in those

children with history of parental myopia; –0.22 D per year if both were myopic vs

–0.07 D per year if one parent was myopic and only –0.02 D per year in those

children with no parental history of myopia.

Strong evidence for the impact of heredity on myopia comes from studies of the

refractive errors of twins. A dizygotic twin study has shown there is significant

linkage to myopia on chromosomes 11p13, 3q26, 8q23 and 4q12 (Hammond et al.,

2004). Furthermore, there is a stronger similarity in the refractive error of

monozygotic than dizygotic twins (Hammond et al., 2001; Lyhne et al., 2001).

The development of sophisticated genetic analysis techniques has established that

there are many genetic loci with linkages to very high forms of myopia, i.e. loci 18q

(Lam et al., 2003; Young et al., 1998), 12q (Young et al., 1998), 4q (Zhang et al.,

2005), 5q (Lam et al., 2008; Ma et al., 2010) and 17q (Paluru et al., 2003). There is

also evidence for a primary genetic basis for moderate forms of myopia: paper

investigating 44 linkage families indicated the locus on 22q12 has an effect on

Literature Review

8

common myopia (Stambolian et al., 2004).

1.2.2 Environmental risk factors for myopia

A number of studies have shown that myopia prevalence is high in groups of people

with high education levels (Wensor et al., 1999; Wu et al., 2001; Xu et al., 2005). For

example, 15,059 Singapore military males aged 16-25 years underwent an

ophthalmic examination and the refraction data were analysed. Within each racial

grouping, the myopia prevalence was positively correlated to education level; 64.1%

with primary school education were myopic compared to 85.0% with a tertiary

education (Wu et al., 2001). Another study randomly selecting subjects from Victoria

in Australia found 12.3% myopic prevalence rate among people who did not

complete secondary education, 20.7% among people with secondary education and

29.5% among people with university education (Wensor et al., 1999).

Similarly, occupations with high nearwork demands also tend to have associated high

myopia prevalence. For example, there are reports of 90% prevalence of myopia

amongst textile workers with a close working distance (~30cm), and few to no

myopes amongst those with sales and production jobs in the same factory (Simensen

& Thorud, 2009). There are reports of 87% of Hong Kong Chinese microscopists

having myopia (Ting et al., 2004), whereas only 23% of Hong Kong fishermen are

myopic (Wong et al., 1993).

Nearwork appears to be a risk factor that is consistently associated with the

development of myopia (Saw et al., 2002; Simensen & Thorud, 2009; Ting et al.,

2004). A study of 1005 school children in Singapore reported that young children

who performed high amounts of reading (e.g. more than two books per week) are

likely to be myopic (Saw et al., 2002). Another study conducted in Xiamen China

suggested that children who spent more hours on nearwork (average 2.2 hours on

reading or writing) in the city have higher myopia prevalence than children who

spent less hours on nearwork in the countryside (Saw et al., 2001).

Literature Review

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1.2.3 Outdoor activity decreases myopia risk

The myopiagenic effect of nearwork appears to be negated by periods spent outdoors.

A cross-sectional study conducting among 2367 school children in Sydney assessed

the relationship between outdoor activity and myopia. They found children who

performed more outdoor activity tended to have a relatively more hyperopic

refraction (Rose et al., 2008). Outdoor activity (≥2 hours per day) reduces the risk of

myopia even if both parents are myopic (Jones et al., 2007). A number of theories

have been proposed as to why this effect has been observed: i) increased general

activity and exercise while outdoors, ii) low accommodation requirements outdoors,

iii) exposure to a greater range of wavelengths outdoors, iv) the extremely high light

levels outdoors compared with indoors.

i) It may be supposed that physical activity induced biochemical changes within the

body that may influence the growth of the eye. A two year longitudinal study of 156

university students found that the amount of physical exercise performed was

inversely correlated to myopia progression, i.e. exercise was inhibitory (Jacobsen et

al., 2008). However, the authors stated that as the physical activity was primarily

conducted outdoors, e.g. bicycle riding outside, it was also possible that the results

were a simply reflection of the amount of time spent outdoors.

ii) It has been suggested that the long viewing distance while outdoors and thus the

low accommodation demand is the reason for the protective effect of outdoor activity.

Constant and high accommodation has long been thought to play a role in the

development of myopia. One theory is that inaccurate accommodation responses

induce a retinal defocus that can be detected by the retina, and the retina passes

signals to the eye to alter the normal eye growth, thus leading to myopia (Flitcroft,

1998).

iii) It has been supposed that the reason why outdoor activity inhibits the

development of myopia is that natural sun light contains a broader wavelength

spectrum than artificial light. The wavelength of outdoor natural light is different

from that of indoor artificial light. Indoor artificial lighting (either incandescent or

Literature Review

10

fluorescent) does not contain UV (ultraviolet radiation) with a reduced spectrum of

approximately 400-700 nm, while the spectrum of sunlight is 290-830 nm

(Thorington, 1985). The possible role of UV (290-400 nm) light in preventing the

development of myopia requires further investigations.

Whether the presence of UV is required to prevent myopia and how UV could be

used by an eye growth system is yet to be determined. One suggestion is that UV

could strengthen the sclera and thus control the axial growth of the eye. A study

investigated a UVA (Ultraviolet A) (315-400 nm) treatment on rabbit eyes in vivo

and showed that the treated biomechanical stress increased 228% (Wollensak et al.,

2005), which means the UVA treatment induced an improvement of the sclera

mechanical strength.

iv) Recent findings suggest that it is simply exposure to extremely high light levels

that is important; both sunlight and bright indoor light inhibit the myopic eye growth

in animal models (Ashby et al., 2009; Ashby & Schaeffel, 2010). When chicks were

reared under two different illuminance levels of artificial lighting, 15000 lux and 500

lux, less form-deprivation myopia was induced in those chicks reared under the

brighter light (Ashby & Schaeffel, 2010). Similarly, short periods of normal vision

are more effective at retarding myopia development if the light level is high and both

sunlight and intense artificial light retarded deprivation myopia (Ashby et al., 2009).

In further support of this idea, progression rates of myopia are less in summer when

light levels are high than in winter, although the long school holidays are typically in

summer (study of 71 school children across the seasons) (Fulk et al., 2002).

The beneficial effect of outdoor activity is thought to be mediated by the retina with

high light levels increasing the release of dopamine (Brainard & Morgan, 1987;

Godley & Wurtman, 1988). Dopamine release is reduced in deprivation myopia in

chickens (Stone et al., 1989; Weiss & Schaeffel, 1993) and monkeys (Iuvone et al.,

1989). Furthermore, the fact that dopamine agonists inhibit myopia in animal models

reveals a regulating role of dopamine in the myopic growth process (Iuvone et al.,

1991; McCarthy et al., 2007; Schmid & Wildsoet, 2004). Injecting dopamine

Literature Review

11

agonists, i.e. apomorphine (Iuvone et al., 1991; Rohrer et al., 1993; Schmid &

Wildsoet, 2004) and quinpirole (McCarthy et al., 2007), inhibits the development of

experimental myopia in chicks.

1.3 Retinal control of eye growth The retina is the primary controller of eye growth with limited involvement from

higher brain centres (Wildsoet & Wallman, 1995). There are three main points of

evidence for this: i) The use of partial diffusers or negative lenses produces localised

regional myopia. Hemifield-lenses when applied over either chick (Diether &

Schaeffel, 1997) or monkey eyes (Smith et al., 2009) induce elongation only in the

treated hemiretina. ii) FDM (Form Deprivation Myopia) and recovery from myopia

occur after the optic nerve, and thus connection to the brain, has been severed (Troilo

et al., 1987). Adaptation to positive and negative spectacle lenses also occurs

following optic nerve section but the endpoint of the emmetropization systems

appears to undergo a hyperopic shift (Wildsoet, 2003). iii) Toxins that destroy the

ganglion cells and prevent the transmission of signals from the retina do not prevent

emmetropization (McBrien et al., 1995; Norton et al., 1994).

1.3.1 Role of inner retina

The involvement of the inner retina in myopia progression has been indicated by

electrophysiological studies. OPs (Oscillatory potentials) which are thought to arise

from the amacrine cells and probably the bipolar cells in the inner retina

(Wachtmeister, 1998) have been investigated in electrophysiological studies. In

chicken experiments, the amplitudes of OPs decreased in form deprivation myopia

(Fujikado et al., 1996; Fujikado et al., 1997). In a human study, OPs displayed

significantly shorter implicit times of the first-order oscillatory potentials in

progressing myopes than in stable myopes and emmetropes (Chen et al., 2006).

These alterations of OPs suggested that the function of the inner retina is changed in

the development of myopia.

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12

Biochemical studies demonstrated that the nuclear protein ZENK (Zif268 Egr-1

NGFI-A Krox-24) (Fischer et al., 1999), Pax-6 (Bhat et al., 2004) and dopamine

(Stone et al., 1989), which are mainly synthesized from the inner retina, have

important roles in regulation of eye growth. ZENK was reduced in both bipolar cells

and amacrine cells in FDM chicks (Fischer et al., 1999). The expression of Pax-6 (a

gene protein) in the retina of FDM chick eyes decreased by day 17 and increased by

day 31 (Bhat et al., 2004). Dopamine release is reduced in the retina of myopic eyes

of FDM chickens (Stone et al., 1989).

1.3.2 Role of outer retina

The outer retina contains the cell bodies of the cones and rods and has a critical role

in ocular growth regulation (Beresford et al., 1998; Liang et al., 1995; Shih et al.,

1993). The cones contain three types of photopigments, with relatively greater

sensitivity to light of long wavelengths, medium wavelengths and short wavelengths.

Therefore the cones are referred to as L cones, M cones and S cones, respectively.

Cones are responsible for colour vision at photopic light levels. The rods contain

only one type of photopigment and are more sensitive than cones. Rods respond to

very low levels of light so that they provide scotopic and mesopic vision (rods and

cones activated) and they have little contribution to colour vision (Cao et al., 2008;

Pokorny et al., 2006). As visual signals arise from photoreceptors and visual

experience is involved in the eye growth (Rabin et al., 1981), investigations of

photoreceptors are necessary for understanding the development of refractive error.

Abnormal structures of photoreceptors in chicks with FDM have been observed with

electron microscopy. In cones, the outer segments are damaged and the inner

segments are thickened. In rods, the outer segments become extremely extended and

thicker (Liang et al., 1995). Another study of myopia in the chick found that both

form deprivation myopia and LIM (Lens-induced Myopia) caused enlargement of

photoreceptor inner segments (Beresford et al., 1998). The significant structural

changes of the outer retina might provide an explanation of the mechanism of FDM.

It has been suggested that the elongation of the photoreceptor outer segments of

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13

FDM chick eyes place pressure on the basal lamina of the RPE (Retinal Pigment

Epithelium), thinning the choroid and collapsing the choroidal vessels, thus choroidal

blood flow is reduced, and the reduction of blood flow could result in myopia

progression (Shih et al., 1993).

1.3.3 Role of RPE

The retinal pigment epithelium lies between the neural retina and the choroid. Visual

signals are detected by photoreceptors, then the eye generates chemical signals which

must go through the RPE layer and then act on the targets-choroid and sclera, thus

the RPE must play an important role in relaying retinal growth signals. Anatomical

studies have found expansion of RPE in FDM chicks (Lin et al., 1993) and

significant topographical changes of RPE in FDM quokka (Fleming et al., 1997;

Harman et al., 1999). The changes to the RPE observed in FDM chickens were

reversible if eyes were allowed to recover from FDM and this recovery happened

first in the retina, and then the RPE and choroid, suggesting that the physiological

functions of FDM were re-established and that molecular signals across the RPE

were used for eye growth regulation (Liang et al., 2004).

1.3.4 Role of choroid

The choroid is the vascular layer between the retina RPE and the sclera and changes

to the choroid have been found during LIM, LIH (Lens-induced Hyperopia) and

FDM and its recovery. If chicks wear positive or negative lenses to impose myopic

or hyperopic eye growth signals, the choroid expands or thins, respectively, to move

the retina forward or backward in order to make the new optical focus fall on the

retina (Wallman et al., 1995). In an FDM study, the eyeball of chicks rapidly

elongated and the choroid thinned following a period of form deprivation; conversely,

during recovery from FDM, the choroid thickened, moving the retina towards the

new plane of optical focus (Fitzgerald et al., 2002).

How the thickness changes of the choroid are mediated is not known. One hypothesis

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14

is that nitric oxide plays a role because intravitreal injection of the nitric oxide

inhibitor L-NAME (NG-nitro-L-arginine Methyl Ester) inhibited choroidal

thickening in both FDM and LIM in chicks (Nickla & Wildsoet, 2004). Another

hypothesis is that choroidal blood flow regulates the thickness of the choroid by

transporting fluid into and out of the choroid (Fitzgerald et al., 2002).

1.3.5 Neurotransmitters in ocular growth

The molecular signals including dopamine, acetylcholine and VIP (Vasoactive

Intestinal Peptide) have been investigated in ocular growth regulation by many

studies. It has been shown that dopamine agonists inhibit myopia in animal models

(Iuvone et al., 1991; McCarthy et al., 2007; Schmid & Wildsoet, 2004), and

dopamine release is reduced in deprivation myopia in chickens (Stone et al., 1989;

Weiss & Schaeffel, 1993) and monkeys (Iuvone et al., 1989). An acetylcholine

antagonist, atropine, inhibited the development of experimental myopia in chicks

when injected intravitreally or subconjunctivally (McBrien et al., 1993; Stone et al.,

1991). VIP antagonists also have inhibit the myopia produced by deprivation (Pickett

Seltner & Stell, 1995).

1.3.6 Role of peripheral retina

Studies of the peripheral retina have documented a link between the pattern of

peripheral refractive errors and degree of myopia. The refractive status of the

peripheral retina relative to the fovea is more hyperopic in myopes and more myopic

in hyperopes than in emmetropes (Charman, 2005; Mutti et al., 2007; Seidemann et

al., 2002; Stone & Flitcroft, 2004). An explanation proposed to account for these

findings is that the on axis myopia is the result of the compensation for the hyperopic

peripheral refractive error, i.e. the peripheral retina elongates and the central retina

follows, thus the off-axis errors induced a change in eye growth that eventually

resulted in the on-axis error.

However it is difficult to say if the central retina or the peripheral retina dominates

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15

the overall axial growth signal. Infant monkeys wearing diffusers with central holes

developed axial elongations (Smith et al., 2005), but chickens wearing lenses with

central holes retain emmetropic axial refraction (Schippert & Schaeffel, 2006). The

former study suggested that visual signals from larger peripheral retina areas could

overshadow the signals from the smaller fovea area, but the latter study suggested

that the foveal retina which has a far greater number of cones than the peripheral

retina plays a critical role in eye growth.

1.4 Photoreceptors and Myopia Visual experience can regulate eye growth (Lauber & Kinnear, 1979). Therefore the

photoreceptors which convert the light signal into a neural signal are likely to be

involved in some way in generating the signals required for eye growth processes.

Evidence for this includes that cone disease usually accompanies refractive error and

the anatomical changes of photoreceptors in experimental myopia in animal models

(Beresford et al., 1998; Michaelides et al., 2005).

1.4.1 Distributions of the Cones and rods

The distributions of the cones and the rods in the retina are different. There are the

highest density of cones in the fovea area, and the density of the cones decreases

sharply when moving away from the fovea. The density of the rods rises from the

fovea to a high value along an elliptical ring at approximately the eccentricity as the

centre of the optic disk and then decreases steadily across the peripheral retina

(Curcio et al., 2004).

1.4.2 Cone disease and refractive error

Cone disease is usually associated with refractive error (Michaelides et al., 2005;

Schwartz et al., 1990). The most important function of cones is to capture photons

and then generate visual signals through a process called phototransduction. If this

function is abnormal or absent, e.g. due to cone disease, the retina without visual

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16

signals will regulate eye growth in an abnormal way. Table 1.2 lists the

characteristics of some cone diseases and the typical refractive errors observed with

each.

TABLE 1.2 Characteristics of some cone diseases.

Cone disease Mode of

inheritance

Refractive

error

Colour vision Fundi

Complete

achromatopsia

Autosomal

recessive

Often

hypermetropia

Absent Usually

normal

Incomplete

achromatopsia

Autosomal

recessive

Often

hypermetropia

Residual

normal

Usually

normal

Oligocone

trichromacy

Autosomal

recessive

Equal

incidence of

myopia and

hypermetropia

Normal Normal

Blue cone

monochromatism

X linked Often myopia Residual tritan

discrimination

Usually

normal

Bornholm eye

disease

X linked Moderate to

high myopia

with

astigmatism

Deuteranopia Myopic

degeneration

1.4.3 Cone involvement in eye growth

The observed anatomical changes of the photoreceptor layer that occur in

experimental myopia in animal models indicates that cones are involved in ocular

growth (Beresford et al., 1998; Liang et al., 1995). The abnormal structure of

photoreceptors in chicks with FDM has been observed via electron microscopy. The

cone outer segments are damaged and the cone inner segments are thickened, and

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17

similarly the rod outer segments become extremely extended and thicker (Liang et al.,

1995). Another study in chickens found that both FDM and LIM caused enlargement

of the photoreceptor inner segments (Beresford et al., 1998).

The involvement of cones in eye growth processes is also shown in retina function

studies (Chen et al., 2006; Chen et al., 2006; Kawabata & Adachi-Usami, 1997;

Kawabata & Adachi-Usami, 1997). An mfERG (Multifocal Electroretinogram) study

in humans found that amplitudes were reduced and the latencies are delayed in

myopia. The authors concluded that the reduced response sensitivity of mfERG in

myopia results primarily from cone function loss (Kawabata & Adachi-Usami, 1997).

In a human based study, OPs displayed significantly shorter implicit times in

progressing myopes than in stable myopes and emmetropes (Chen et al., 2006).

Another psychophysical study showed reduced visual sensitivity in high myopes

(Jaworski et al., 2006). Sensitivity to white and S cone isolation spots of various size

and contrast were tested in 10 high myopes and 10 control emmetropes. The myopic

group showed a loss of sensitivity at all spot sizes.

In a study utilising the chick eye growth model, chicks were form deprived with

translucent diffusers and exposed to different light conditions. Both sunlight and

intensely bright indoor room light retarded the progression of deprivation myopia

(Ashby et al., 2009). Moreover, it has also been reported that chicks develop myopia

when raised under dim light conditions (Lauber & Kinnear, 1979). In contrast, cats

which are largely nocturnal and have a rod dominated retina do not develop myopia

when wearing contact lenses to induce a predictable degree of myopia or with lid

suture (Nathan et al., 1984).

1.4.4 Cone ratios in refractive error

Considering the important role of light in eye growth and that photopic vision

predominantly depends on cones, the modulation of refractive error could be led by

cone inputs and outputs. It may be speculated that either the function or the

structures of cones varies between myopic and emmetropic eyes. It has been reported

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18

that a monochromatic yellow light matched by an emmetrope using red and green

seemed too red to a myopic subject (normal correction and near monochromatic

stimuli during experiment) and too green to a hyperopic subject (Wienke, 1960),

suggesting that myopes have greater sensitivity to red and hyperopes to green. This

finding is consistent with another study finding that myopes have relatively higher

sensitivity to long-wavelength light than emmetropes (Rucker & Kruger, 2006). The

participants viewed stimuli with different cone contrast ratios while their

accommodation was measured continuously. Their results indicated that more

myopic individuals showed a higher level of accommodation gain to relatively more

L cone contrast.

As L cones have their peak sensitivity at long wavelengths (567nm) and M cones

have their peak sensitivity at medium wavelengths (541nm), the relatively higher

sensitivity to red in myopia might be postulated to be due to myopes having higher

L/M cone ratios than emmetropes (Rucker & Kruger, 2006). However,

psychophysical evidence indicates that post-receptoral gain control normalizes any

differences in sensitivity to red and green lights (Raleigh match), hence individual

differences in Raleigh matches are small compared to the population variability in

cone ratios (Pokorny et al., 1991).

Although several studies have recorded the L/M ratio in participants with normal

colour vision, there are limited investigations comparing the L/M ratios in myopia

and emmetropia. The L/M cone ratio has been reported to range from 0.4 to 13

among participants with normal colour vision as assessed by various techniques

(Albrecht et al., 2002; Bowmaker & Dartnall, 1980; Brainard et al., 2000; Carroll et

al., 2000; Carroll et al., 2002; Cicerone & Nerger, 1989; Dartnall et al., 1983;

Hagstrom et al., 1998; Hagstrom et al., 2000; Hood et al., 2002; Kremers et al., 1999;

Langrová et al., 2007; Nerger & Cicerone, 1992; Otake & Cicerone, 2000; Pokorny

& Smith, 1987; Roorda & Williams, 1988; Usui et al., 1998).

The L/M cone ratios amongst human individuals with normal colour vision reported

in different studies are shown in Table 1.3. L/M cone ratios range from 1.5 to 4.9

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based on analysis using gene expression and from 1.3 to 1.7 using

microspectrophotometry (Hagstrom et al., 1998; Hagstrom et al., 2000). These two

methods collected donor eyes to analyse the mRNA of L pigment and M pigment of

the retina. Direct images of the retina were taken when the photopigments were

selectively bleached. This method gave L/M ratios ranging from 1.15 to 3.79

(Bowmaker, & Dartnall, 1980; Dartnall et al., 1983; Roorda, & Williams, 1988).

Several psychophysical studies have been carried out and the reported L/M ratios

range from 1.1 to 3.4 (Otake, & Cicerone, 2000; Pokorny, & Smith, 1987; Cicerone,

& Nerger, 1989; Nerger, & Cicerone, 1992). Electrophysiological techniques have

also been used to estimate L/M ratios and values with this method ranging from 0.4

to 13 (Brainard et al., 2000; Usui et al., 1998; Carroll et al., 2002; Carroll et al., 2000;

Kremers et al., 1999; Langrova et al., 2007; Albrecht et al., 2002; Hood et al., 2002).

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TABLE 1.3 Cone ratios amongst people with normal colour vision.

Method Participant

Number

Retinal

area

L/M cone

ratio

Author

mRNA analysis 2 20° and

40°

1.5 (20°)

3.0 (40°)

Hagstrom et

al., 1998

Cone pigment gene

expression

4 20° 3.63 – 4.86 Hagstrom et

al., 2000

Microspectrophotometer 1 Foveal

and

parafov

eal

1.73 Bowmaker,

& Dartnall,

1980

Microspectrophotometer 7 Foveal

and

parafov

eal

1.29 Dartnall et

al., 1983

Combine adaptive optics

and retinal densitometry

2 1° 1.15 - 3.79 Roorda, &

Williams,

1988

Unique yellow 2 28° 1.63 - 1.99 Otake, &

Cicerone,

2000

Heterochromatic flicker

photometry

2 2° 1.1-3.4 Pokorny, &

Smith, 1987

Heterochromatic flicker

photometry

6 1° 1.46 - 2.36 Cicerone, &

Nerger, 1989

Psychophysical

prosedures

3 4° 2.0 Nerger, &

Cicerone,

1992

Flicker 2 1° 1.06 - 3.38 Brainard et

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electroretinogram al., 2000

Flicker

electroretinogram

5 124*10

8°

3.7 ± 1.0 Usui et al.,

1998

Flicker-photometric

electroretinogram

62 70° 0.4 - 13

Carroll et al.,

2002

Flicker-photometric

electroretinogram

15 70° 0.6 - 12

Carroll et al.,

2000

Electroretinogram 36 124*10

8°

4.24 ± 2.13 Kremers et

al., 1999

The multifocal pattern

electroretinogram

10 8° and

12.8°-2

6°

1.00 - 2.78 (8°)

1.29 - 2.78

(12.8°-26°)

Langrova et

al., 2007

Multifocal

electroretinograms

38 5° and

40°

1.4 (5°)

2.3 (40°)

Albrecht et

al., 2002

Multifocal visual

evoked potentials

6 1° and

5.8°-22.

2°

0.8 - 1.1 (1°)

1.1 - 1.7

(5.8°-22.2°)

Hood et al.,

2002

In these L/M cone ratios studies, only three papers mentioned the refraction of the

participants (Table 1.4). In the study of Otake and Cicerone (2000) for the central 28°

using unique yellow, the L/M ratios of one emmetrope and one myope were 1.63 and

1.99. Using heterochromatic flicker photometry and a 1° field, the L/M ratios of four

emmetropes were 2.07, 2.00, 1.46, 2.36, respectively; the L/M ratios of two mild

myopes were 2.28 and 1.99. Finally, using psychophysical procedures and a 4° field,

the L/M ratio of one emmetrope was 1.83; the L/M ratios of two mild myopes were

2.2 and 1.5. These data suggest great individual variability in the L/M cone ratio in

persons with emmetropia and myopia.

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TABLE 1.4 The refraction of participants in three L/M cone ratio studies

Method Retinal

area

Participant

ID

Refraction L/M

cone

ratio

Author

Unique yellow 28° KL

SL

-2.25D

Emmetrope

1.99

1.63

Otake, &

Cicerone,

2000

Heterochromatic

flicker

photometry

1° VV

HA

CC

JN

YP

EM

<-1.50D

<-1.50D

Emmetrope

Emmetrope

Emmetrope

Emmetrope

2.28

1.99

2.07

2.00

1.46

2.36

Cicerone, &

Nerger, 1989

Psychophysical

procedures

4° CC

VV

HA

Emmetrope

<-2.00D

<-2.00D

1.83

2.2

1.5

Nerger, &

Cicerone,

1992

1.5 Methods to estimate L/M cone ratios The L/M cone ratios of human beings have been assessed using different methods.

Analyses of L/M cone ratios using gene expression and microspectrophotometry

have been performed on donor eyes (Hagstrom et al., 1998; Hagstrom et al., 2000).

The mRNA of photoreceptors of selected retina patches was analysed. The ratios of

L/M cone were calculated from the L/M mRNA ratios. Direct images of the retinal

cones in living eyes have been performed however the special device that was used is

not commonly available (Bowmaker & Dartnall, 1980; Dartnall et al., 1983; Roorda

& Williams, 1988). The images were obtained after the L or M cones were fully

bleached. The number of L or M cones can be counted from the direct images.

Psychophysical studies have been conducted using detection thresholds,

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23

heterochromatic flicker photometry and minimal flicker perception (Kremers et al.,

2000). High temporal frequencies and low temporal frequencies of the

psychophysical tasks were compared and the authors found that there is a gain

adjustment to compensate the differences of L/M cone ratios when using low

temporal frequencies which tap into the red-green chromatic channel.

Psychophysical studies are noninvasive, but usually measure the ratio only on the

foveal retina. Electrophysiology methods such as flicker-photometric

electroretinogram, mfERG and mfVEP also have been used to estimate L/M cone

ratios. These measurements are more reliable because they are objective, but one

study using flicker-photometric electroretinogram suggested that the variation of the

sensitivity of photopigments between individuals provide a major source of error in

estimates of L/M cone ratios, but this error usually within about 2% (Carroll et al.,

2000).

1.6 Multifocal visual evoked potential Every technique of cone isolation study has limitations and advantages and all

require assumptions (for example, choice of cone fundamentals, corrections for

pre-receptoral filtering, method for determining isoluminant points) (Hood et al.,

2002) to derive an estimate of the cone ratio from the data. In this study, mfVEP was

recorded with cone isolation stimuli as mfVEP is a non-invasive and widely accepted

technique.

The visual evoked potential (VEP) is an electrical potential recorded from the

primary visual cortex of a human or other animal following presentation of a light

stimulus (Hood et al., 2003). Electrodes are placed on the scalp to record the VEP

signals. There are two electrodes to record VEP signals: an active electrode and an

inactive electrode. The algebraic difference in potential is determined by VEPs

between these two electrodes. The VEPs are used to examine the responses of the

visual pathway from the retina, the optic nerve, optic radiations and occipital cortex.

The VEP is not a new technique in neuro-ophthalmology field. Optic neuritis

http://en.wikipedia.org/wiki/Electrical_potential

http://en.wikipedia.org/wiki/Human

http://en.wikipedia.org/wiki/Animal

http://en.wikipedia.org/wiki/Stimulus_(physiology)

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24

sclerosis had been tested using VEP in patients forty years ago and it reported

delayed VEP responses (Halliday et al., 1972). It has been proved that VEP is

sensitive in detecting visual field defects. One patient with a right homonymous

hemianopia with maular sparing was found an absence of unpatterned light VEP

signals when the right visual field was stimulated (Regan & Cartwright, 1970).

The multifocal visual evoked potential is a type of pattern reversal VEP with inputs

from multiple visual areas (Hood et al., 2003). The standard stimuli are a black-white

dartboard array consisting of 60 sectors and each sector is a checkerboard pattern

consisting of 16 checks. The advantage of mfVEP compare to VEP is the size of the

stimuli used. With smaller stimuli, the mfVEP can easily detect local defects. The

mfVEP has been used to rule out non-organic visual loss (Miele et al., 2000);

diagnose optic neuritis (Hood et al., 2000) and to assess the damage of optic nerve in

glaucoma (Hood et al., 2003). Recently the mfVEP has been used to determine L/M

cone ratios (Hood et al., 2002).

1.7 Colour vision and colour vision in myopia A recent study indicated that red-green colour defective individuals are not usually

myopic or emmetropic (Qian et al., 2009); i.e. they suffer a range of refractive errors.

As the cones in the retina are responsible for colour vision in humans, more studies

are required on the role of cones in the progression of myopia.

1.7.1 Colour vision

Visible light is the limited portion of the radiant energy to which the eye responds.

The visible light spectrum is from 400 nm to 700 nm. The processing of visual

signals in humans begins in three types of photoreceptor which absorb photons of

particular wavelengths. The S-cone, M-cone and L-cone sensitive photopigments

have peak sensitivity in the short (445nm), middle (541nm) and long (567nm)

wavelength region of the spectrum, respectively (Smith & Pokorny, 1975).

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Visual signals are directly transmitted from photoreceptors to the bipolar cells and

ganglion cells. The other visual pathway is from photoreceptors via horizontal cells

and amacrine cells to the bipolar cells and ganglion cells (Lee et al., 2010). Three

major types of ganglion cells are involved in human colour vision: the midget

ganglion cells, the parasol ganglion cells and the small bistratified ganglion cells.

Visual signals are transmitted from the ganglion cells to the visual cortex following

three primary neural pathways. The three neural pathways are named according to

the laminae within the laterale geniculate nucleus (LGN): PC (parvocellular), MC

(magnocellular) and KC (koniocellular) pathways (Dacey, 2000).

The midget ganglion cells target to the parvocellular LGN, while the parasol

ganglion cells project to the magnocellular LGN. The MC pathway is sensitive to

luminance contrast and processes combined signals from L and M cones (Lee et al.,

1988; Perry et al., 1984). The PC pathway is responsible for chromatic detection and

receives opponent signals from L and M cones. The PC pathway is important for the

red-green colour vision (Lee, 1996; Merigan, 1989). The small bistratified ganglion

cells which connected to S cones feed to the KC pathway. The KC pathway belongs

to the short-wavelength system (Dacey, 1993; Dacey, 1996).

Human beings and old world primates are trichromats, but placental mammals are

dichromats (Jacobs, 1993). While primates have an advanced colour vision system,

many vertebrates with four cone pigments developed colour vision better than ours.

In vertebrates, the overlap in sensitivity between adjacent cones was reduced,

therefore the discrimination of colours was increased (Govardovskii & Zueva, 1977;

Loew & Govardovskii, 2001; Palacios et al., 1998). The most sophisticated colour

vision system has at least ten types of photoreceptors founded in mantis shrimps

(Cronin & Marshall, 1989).

The evolution of colour vision in animals probably depends on fruits and flowers. In

late 19th century, the evolution of colour vision was formulated: ‘Insects produce

flowers. Flowers produce the colour-sense in insects. The colour-sense produces a

taste for colour. The taste for colour produces butterflies and brilliant beetles. Birds

Literature Review

26

and mammals produce fruits. Fruits produce a taste for colour in birds and mammals.

The taste for colour produces the external hues of humming-birds, parrots and

monkeys. Man’s frugivorous ancestry produces in him a similar taste and that taste

produces the final result of human chromatic arts.’ (Wallace, 1879).

The benefit of having chromatic vision is to easy detect and distinguish the objects in

the world. As shown in Figure 1.2, with normal colour vision the red flowers are

clear, while without colour perception the red flowers are somewhat vague.

Trichromatic colour vision helps primates to separate green, yellow, orange and red

fruits as fruits are an important diet of primates (Tan & Li, 1999). Also the set of

reddish colour was developed because many primates have red sexual displays

(Dixson, 1999).

Literature Review

27

FIGURE 1.2 Red flowers, green leaves and blue sky with and without colour (This photograph

was taken by Nanyu Zhou). With normal colour vision the red flowers are clear, while without

colour perception the red flowers are somewhat vague.

1.7.2 Colour deficiency and myopia

Recent studies giving insight into colour vision and myopia infer that colour vision

could influence the development of myopia (Kröger & Binder, 2000; Qian et al.,

2009). One study investigated the prevalence of myopia in 309 high school students

with colour vision deficiency, including 142 students with protan and 167 students

with deutan and 927 students with normal colour vision. They concluded that

students with red-green colour vision deficiency have both lower incidence and

lower degrees of myopia than those students with normal colour vision (Qian et al.,

Literature Review

28

2009). Another study investigated the effect of long wavelengths during nearwork.

They used a white paper which was illuminated by a laboratory halogen lamp and

interference filters which selectively absorbed long wavelengths to education

nearwork. The paper significantly reduced accommodation stimulus by about 0.5 D

(Kröger & Binder, 2000). As accommodation is considered as a possible cause of

myopia (Chen et al., 2003), it is reasonable to speculate that the red light absorbing

paper may slow the progression of myopia.

The mechanism of the influence of red and green light in myopia might be mediated

by longitudinal chromatic aberration (LCA) (Qian et al., 2009). White light is a mix

of lights with different wavelengths. Among emmetropes, after white light goes

through the refractive media of the eye, red light is focused behind the retina; yellow

light is focused in the plane of the photoreceptors; green light is focused in front of

the retina. If the retina is more sensitive to red than green light or has higher L cone

sensitivity than M cone sensitivity, the retina could move toward red by elongating

the eyeball or increasing accommodation, resulting in myopia. A recent study has

found that higher myopic individuals have higher mean accommodation levels to

L-cone stimulation. The possible explanation is that myopic individuals have

relatively higher L/M cone ratios or the differences in pigment polymorphism

between myopia and emmetropia (Rucker & Kruger, 2006).

1.8 Aims and hypothesis There has been some investigations of the cone activity in myopia (Kawabata &

Adachi-Usami, 1997) and the cone ratios in the normal colour vision population

(Kremers et al., 2000), but little work comparing the cone ratios between eyes of

myopic and emmetropic individuals with normal colour vision.

Given the fact that dichromats (red-green colour vision deficiency), with extreme

L/M cone ratios, are not all myopic or emmetropic (Qian et al., 2009), we predict

that the L/M cone ratio will vary in individuals but not show a regular difference

simply due to refractive error. On the other hand, Neitz and Neitz (2011) suggested

Literature Review

29

that eyeglasses that equalize the activities of L and M cones can reduced the myopia

progression rate to a tenth of that observed in myopes wearing standard eyeglasses;

this suggests that the L and M cone ratios may be different in myopes and

emmetropes.

The aim of this research was to determine if L/M cone ratios in the central retina are

different between myopic and emmetropic young, colour normal adults. The null

hypothesis was that myopia is not dependent on L/M cone ratio. The alternate

hypothesis was that myopia is caused by a high L/M cone ratio.

Experimental Methods

30

Chapter 2


2.1 Introduction This chapter describes the mfVEP technique in detail, the calibration of the monitors

and the calculation of L and M cone isolating stimuli for studying the L/M cone

ratios.

2.2 Multifocal visual evoked potential for L/M cone ratio study Figure 2.1 shows the mfVEP equipment and stimuli used in this study, including a

CRT stimulator, an amplifier and a Apple Mac G5 control computer. With the mfVEP,

multiple responses can be tested from different locations of the retina. Electrodes

were connected to the amplifier and placed on the scalp to detect the electrical

signals generated by the retina and the visual nervous system. VERIS system was

used to generate visual stimuli and collect data. The L/M cone ratio can be calculated

from mfVEP responses (Hood et al., 2002).


31

FIGURE 2.1 The mfVEP equipment and stimuli used in this study, including a CRT stimulator,

an amplifier and a mac control computer. During experiments, participants sat in front of the

monitor and looked at the stimuli. Some electrodes which connected to the amplifier were placed

on their scalp to detect the electrical signals generated by the retina and the visual nervous system.

VERIS system was used to generate visual stimuli and collect data.

The specific stimulator parameters of the mfVEP are shown in Table 2.1. The target

for participants to fixate is a cross with diameter 2°. The pen size of the fixation

means the thickness of the cross. Kernel slice is the response to a particular stimulus

pattern. In temporal settings, max kernel order is 3 and max kernel spread is 4.

STIMULATOR

MAC CONTRAL COMPUTER

AMPLIFIER


32

TABLE 2.1 The specific stimulator parameters of the mfVEP

GEOMETRY Screen Distance: 37cm

Height: 20cm

Width: 20cm

Fixation Type: cross

Diameter: 2°

Pen size: 9

Fixation x: 0

Fixation y: 0

TEMPORAL Frame rate: 75 Hz

M-sequence exponent: 15

Frames per m-step: 1

Max kernel order: 3

Max kernel spread: 4

Memory: <320 ms

No. of segments: 16

Samples per frame: 16

Pre-exposure: 1000 ms

AQUISITION Board type: PCI 1200

Analogue channels: 1

Board gain: 1

External amplifier: grass 15 LT

Gain: 100 K

Low cutoff: 3 Hz

High cutoff: 100 Hz

Notch filter: out


33

According to the ISCEV (International Society for Clinical Electrophysiology of

Vision) standard for clinical visual evoked potentials, a single black-white

pattern-reversal VEP response consists of N75, P100 and N135 peaks. The N and P

stand for negative and positive peak and the numbers 75, 100 and 135 represent

recording time (Odom et al., 2010)(see Figure 2.2).

FIGURE 2.2 A single black-white pattern-reversal VEP response. Typically it consists of N75,

P100 and N135 peaks (Odom et al., 2010). The N and P stand for negative and positive peak and

the numbers 75, 100 and 135 represent recording time.

The amplitudes of mfVEPs are highly variable among individuals, but the repeat

reliability of mfVEP responses is good ( the standard deviation of the root mean square

measure amplitude is approximately 1.6dB) for the same subject) (Chen et al., 2003).

L/M ratios have been studied using mfVEP by Hood et al. (2002). The L/M ratios are

calculated from the N1-P1 amplitudes in our study (Figure 2.3). N1 was the first

negative peak and P1 was the first positive peak. To improve the variation of mfVEP

recordings within individuals, two same mfVEP measurements were averaged.

Time

Amplitude


34

FIGURE 2.3 An example that how to calculate L/M cone ratios in this study. The L/M cone ratios

were calculated from the N1-P1 amplitude. N1 is the first negative peak of the mfVEP response

and P1 is the first positive peak. The red curve is the mfVEP signal of L modulation and green

curve is the mfVEP signal of M modulation. In this example, the N1-P1 amplitude of L

modulation is 0.382 µV and M modulation is 0.354 µV. The L/M ratio is calculated as

0.382/0.354=1.08.

2.3 Calibration Both CRT (Cathode Ray Tube) (Gateway EV500A) and LCD (Liquid Crystal

Display) (Viewsonic VX922) monitors were available to present mfVEP stimuli in

our laboratory. The frame rate of CRT and LCD are both 75 Hz (see section 4.5). The

stimuli from CRT and LCD were driven by the Visual Evoked Response Imaging

System (VERIS 5.1, Electro-Diagnostic Imaging Inc., San Mateo, CA, USA). To

determine whether it was better to use a CRT or an LCD, we conducted a spectral

and luminance calibration on both monitors. The results showed CRT was better than

LCD, because the phosphor response of CRT is faster and the luminance output of

the CRT is greater. A further calibration of gamma correction was performed on CRT

L modulation N1-P1 amplitude

M modulation N1-P1 amplitude


35

in order to accurately control the outputs of the primary lights.

2.3.1 Spectral and luminance calibration of CRT and LCD monitors

In this study, we used alternating coloured lights to stimulate only one cone type, and

therefore the phosphor response, phosphor output and luminance output of the

monitors used is important. Quick phosphor responses allow colour lights to change

more accurately across short time intervals. A narrow phosphor output can provide a

larger contrast range for isolating the cones. A greater luminance output range as a

function of the output voltage of the video card bit number can give a larger range of

cone excitations and thus are problematic for designing stimuli to isolate different

cone types.

The effects of the CRT and LCD phosphor response on mfERG recording have been

studied by Feigl and Zele (2008). The mfERG recording used the VERIS system

with the CRT and was repeated with the LCD. They showed that when an LCD

monitor was used as a stimulus generator, there was a delay in the peak implicit time

of the mfERG waveforms relative to those when a CRT monitor was used.

The emission spectra of red, green and blue phosphors at maximum output (bit value

at 255) of the CRT and the LCD were measured using a calibrated Spectroradiometer

(Tampa, Florida, USA). Figure 2.4 shows the spectral distribution of the three

phosphors. The CRT and LCD both have a similar wavelength range, but the

emission spectral of the green phosphor of LCD is narrower.


36

FIGURE 2.4 The spectral distribution of red, green and blue from the CRT and the LCD

monitors. The CRT and LCD both have a similar wavelength range, but the emission spectral of

the green phosphor of LCD is narrower.


37

The luminance outputs of the CRT and the LCD were measured using a luminance

colorimeter (BM-7, Topcon, Tokyo, Japan). Each colour was measured at bit

numbers from 1 to 255. Figure 2.5 shows the luminance output of the CRT and the

LCD monitor driven by the VERIS system. The luminance output of the CRT and

LCD becomes flat at low and high bit values. The effective bit number ranges of

CRT are 49-255 for red, 27-255 for green and 99-255 for blue. The bit ranges of

LCD are 59-212 for red, 32-214 for green and 104-218 for blue, but the maximum

luminance output of each phosphor is higher than for the CRT. The reduced bit range

is due to the VERIS system driving the display (the CRT or LCD display). Although

it has an 8 bit video card with a full 255 bit range, the display output clearly has

lower and upper limits that are less than 8 bit at both a high range and a low range.


38

FIGURE 2.5 The luminance output of CRT and LCD monitors driven by the VERIS system. The

luminance output of the CRT and LCD becomes flat at low and high bit values. The effective bit

number ranges of CRT are 49-255 for red, 27-255 for green and 99-255 for blue. The bit ranges

of LCD are 59-212 for red, 32-214 for green and 104-218 for blue, but the maximum luminance

output of each phosphor is higher than for the CRT.


39

Although the LCD monitor has narrower green phosphor output than the CRT, the

temporal phosphor response of the CRT has a faster rise time and the luminance

output of the CRT is higher than those of the LCD. Thus, in this study, we used the

CRT monitor to generate the cone isolating mfVEP stimuli.

2.3.2 Gamma correction of CRT monitor

The luminance output of the CRT was measured using a luminance colorimeter

(BM-7, Topcon, Tokyo, Japan). The VERIS system controlled the RGB bit numbers

from 1 to 255. Appendix I is the original luminance outputs from the CRT monitor.

Because the luminance of a CRT changes over a period of time, the monitor needs to

be warmed up for about 1 hour before measurements (Metha et al., 1993).

An ideal display behaves linearly, but the CRT monitor is nonlinear and the

luminance output is not linearly related to the voltage input. In fact, the relationship

between the voltage input and luminance output of CRT monitor is subject to a

power function. Such a relationship is known as "gamma function" and described by

the following equation:

Lout = α + βLin γ (1)

where Lout is the luminance output, Lin is the voltage input, α is the dark light of the

monitor, β is a constant and γ is the exponent of Lin.

We fitted gamma functions to the red, green and blue in the effective range,

respectively. Figure 2.6 shows the relative voltage-luminance relationship (gamma

function) of the CRT monitor. The gamma is usually between 2-3 for CRT (Colombo

& Derrington, 2001). The CRT in our laboratory has the gamma exponent of 2.16 for

red, 2.02 for green and 2.45 for blue. The voltage-luminance relationship for the blue

phosphor has some flats, shown in Figure 2.6. Given that the blue phosphor does not

have large modulation amplitude for the L and M cone isolations (see section 2.4.1),

thus it will have minimum impact on the outcome.


40


41

FIGURE 2.6 The modelled gamma functions of our CRT monitor. The gamma value is 2.1624

for red, 2.0183 for green and 2.4507 for blue. There are some flats of the voltage-luminance

relationship for the blue phosphor. Given that the blue phosphor does not have a large modulation

amplitude for the L and M cone isolations, thus it will have minimum impact on the outcome.

In order to obtain a linear voltage-luminance relationship, gamma correction is

required to compensate the original data. To achieve gamma correction, an inverse

function is generated:

Lout' = [(Lout - α)/β]1/γ (2)

where Lout' is the desired output, and α, β, γ and Lout are as defined in equation (1).

The principle of gamma correction is shown in Figure 2.7 using the example of the

green gun.


42

FIGURE 2.7 Gamma function and its inverse function for the green gun. The lower curve is the

modelled gamma function and the top curve is the inverse function of the model. A relative input

voltage of 0.6 (A) in the modelled gamma function results in 0.36 (B). With a linear relationship,

the input 0.6 should result in output 0.6. The nonlinear relationship can be corrected by applying

an inverse function. When the input 0.6 is entered, a value of 0.77 (C) is returned. Appling the

value 0.77 to the relative input voltage would return the required output of 0.6.

With the nonlinear input-output relationship, a relative input voltage of 0.6 (A)

results in 0.36 (B). With a linear relationship, the input 0.6 should result in output 0.6.

The nonlinear relationship can be corrected by applying an inverse function. When

the input 0.6 is entered, a value of 0.77 (C) is returned. Appling the value 0.77 to the

relative input voltage would return the required output of 0.6.

As shown in Figure 2.8, the input-output relationship became linear after the gamma

correction had been made. To evaluate the linear relationship, we computed if the

maximum luminance plus the minimum luminance equals twice the average

luminance. The error of the linearization check is -0.024% for red, -0.017% for green

and -0.007% for blue, which are minor (see Appendix II).


43


44

FIGURE 2.8 Input-output relationship of the red, green and blue guns after the gamma correction

had been finished. To evaluate the linear relationship, we computed if the maximum luminance

plus the minimum luminance equals twice the average luminance. The error of the linearization

check is -0.024% for red, -0.017% for green and -0.007% for blue.

2.4 Calculation of L and M cone isolation For isolating only one type of cone, we used a silent substitution technique (Estevez

& Spekreijse, 1982). We used two conditions. In the first condition, we generated L

cone excitation and silenced M and S cones. In the second condition, we generated

M cone excitation and silenced L and S cones. The rods excitation was minimized by

the background room lights (approximately 100 cd/m2) and flickering lights of

mfVEP. All the calculations were performed using Excel spreadsheets.

2.4.1 Silent substitution

The principle of the silent substitution technique is that if alternating between two

lights with physically different spectral distributions, but that produce equal


45

excitation of one type cone, this photoreceptor should not be able to detect the

change in wavelength of the lights. As shown in Figure 2.9, the 543nm and 594 nm

lights give the same excitation to the L cones, but different excitation to the M cones.

When alternating between 543nm and 594 nm, the L cones cannot detect the

difference, so the responses of the M cones are isolated (Foster, 2010).

FIGURE 2.9 An example of the principle of the silent substitution technique. The 543nm and 594

nm lights give the same excitation to the L cones, but different excitation to the M cones. When

alternating between 543nm and 594 nm, the L cones cannot detect the difference, so the

responses of the M cones are isolated.

The excitation of one type of cone can be controlled by modulating the output of

three primary lights such as the RGB phosphors in a CRT by multiplying the

emission spectra of the three phosphors (see Appendix IV) with the cone

fundamentals and integrating the product over wavelength. In this study the Smith

and Pokony 10° cone fundamentals (see Appendix V) was employed. Figure 2.10

shows the sensitivity of each photoreceptor type according to the Smith and Pokony

10° fundamentals (Smith & Pokorny, 1975).


46

FIGURE 2.10 The Smith and Pokony 10° fundamentals (Smith & Pokorny, 1975). The red, green

and blue curves are the L cone, M cone and S cone sensitivity over 400-700nm wavelengths,

respectively.

For example, the L cone excitation by a generated red phosphor is:

LR = ∫ ER * FL * d(λ) (3)

where ER is the emission distribution of the red phosphor and FL is the L cone

fundamental. Similarly the L cone excitation by generated green phosphor is:

LG = ∫ EG * FL * d(λ) (4)

the L cone excitation by generated blue phosphor is:

LB= ∫ EB * FL * d(λ) (5)

400

700

700

700

400

400


47

the M cone excitation by generated red phosphor is:

MR = ∫ ER * FM * d(λ) (6)

the M cone excitation by generated green phosphor is:

MG = ∫ EG * FM * d(λ) (7)

the M cone excitation by generated blue phosphor is:

MB = ∫ EB * FM * d(λ) (8)

the S cone excitation by generated red phosphor is:

SR = ∫ ER * FS * d(λ) (9)

the S cone excitation by generated green phosphor is:

SG = ∫ EG* FS * d(λ) (10)

the S cone excitation by generated blue phosphor is:

SB = ∫ EB * FS * d(λ) (11)

By applying equation (3) - (11), we can obtain a 3*3 matrix:

R G B L 65.61829087 153.3281353 24.49526207 M 15.06004851 93.94170552 22.24061625 S 13.50035923 45.72929708 388.9271096

700

700

700

700

700

700

400

400

400

400

400

400


48

Since every light contains three primaries R, G and B with a certain scale, the total

excitation of the L, M and S cones can be calculated:

The R, G and B output value can be calculated using the inverse matrix of the above

3*3 matrix:

The RGB and LMS can be modulated by some value ΔR, ΔG, ΔB, ΔL, ΔM and ΔS:

If we make ΔM = 0 and ΔS = 0, only the L cone is excited. Similarly only the M cone

is excited when ΔL = 0 and ΔS =0 and only the S cone is excited when ΔL = 0 and

ΔM = 0.

2.4.2 The L cone modulation

The L cone modulation was calculated using an Excel spreadsheet. Making ΔL =

ΔLmax, ΔM = 0 and ΔS = 0 resulted in ΔR = 1, ΔG = -0.1565 and ΔB = -0.0163. The

ΔR, ΔG and ΔB correspond to percentages and the “-” means decrease. We

normalized the available ranges of bit numbers in Appendix III, thus the two lights

can be selected from Appendix I, II and III. For example, according to Appendix III,

the bit number for ΔR = 1 are from 0 to 255, then from Appendix II, in a linear

ΔR

ΔG

ΔB

ΔL

ΔM

ΔS

= *

0.024253911 -0.039954945 0.000757254

-0.003794518 0.017200641 -0.000744626

-0.000395745 -0.00063551 0.002632442

65.61829084 153.3281353 24.49526207

15.06004851 93.94170552 22.24061625

13.50035923 45.72929708 388.9271096

R

G

B

L

M

S

= *

0.024253911 -0.039954945 0.000757254

-0.003794518 0.017200641 -0.000744626

-0.000395745 -0.00063551 0.002632442

= *

R

G

B

L

M

S


49

relationship, the luminance of red correspond to 255 is 36.729 cd/m2. The real

luminance output is not linear, so we need to find the original bit number giving

luminance output 36.729 cd/m2, which is 243 from Appendix I. Table 2.2 has the

RGB values applied to the RGB phosphors in this study to generate lights that

stimulate only L cones.

TABLE 2.2 The RGB values of the L cone modulation

Light 1 Light 2

R 243 0

G 0 122

B 0 176

The L cone contrast was calculated using the Michelson contrasts equation:

( Lmax – Lmin ) / ( Lmax + Lmin ) * 100% (12)

in which the Lmax and Lmin are the maximal and minimal L cone excitation,

respectively. In this study, Light 1 gives the maximal L cone excitation and Light 2

gives the minimal L cone excitation.

The maximum L cone contrast is:

[(1*65.61829084 + 0*153.3281353 + 0*24.49526207) – (0*65.61829084 +

0.1565*153.3281353 + 0.0163*24.49526207)] / [(1*65.61829084 + 0*153.3281353

+ 0*24.49526207) + (0*65.61829084 + 0.1565*153.3281353 +

0.0163*24.49526207)] * 100% = 45.8 %


50

2.4.3 The M cone modulation

The M cone modulation was calculated using an Excel spreadsheet. Making ΔL = 0,

ΔM = ΔMmax and ΔS = 0 resulted in ΔR = -1, ΔG = 0.4305 and ΔB = -0.0159. The

ΔR, ΔG and ΔB corresponded to percentage and the “-” means decrease. The two

lights can be selected form Appendix I, II and III. Table 2.3 is the stimulus applied in

this study to stimulate only M cone:

TABLE 2.3 The RGB values of the M cone modulation

Light 1 Light 2

R 0 243

G 177 0

B 0 176

The M cone contrast was calculated using the Michelson contrasts equation:

( Mmax – Mmin ) / ( Mmax + Mmin ) * 100% (13)

in which the Mmax and Mmin are the maximal and minimal M cone excitation,

respectively. In this study, Light 1 gives the maximal M cone excitation and Light 2

gives the minimal M cone excitation.

The maximum M cone contrast is:

[(0*15.06004851 + 0.4305*93.94170552 + 0*22.24061625) - (1*15.06004851 +

0*93.94170552 + 0.0159*22.24061625)] / [(0*15.06004851 + 0.4305*93.94170552

+ 0*22.24061625) + (1*15.06004851 + 0*93.94170552 + 0.0159*22.24061625)] *

100% = 44.8 %


51

2.5 Statistical analysis

SPSS (Statistical Package for the Social Sciences, 18.0) was used to conduct the

statistical analysis. One-way ANOVA was performed to determine whether there are

differences in L/M cone ratios between emmetropes, lower myopes and higher

myopes. Independent-samples t-test was performed to determine whether there are

differences in L/M cone ratios between emmetropes and myopes.

2.6 Power analysis

GPower (3.1.5) software was used to conduct the power analysis. Priori power

analysis was performed to estimate sample size. A previous L/M cone ratios study

was used to predict the sample size of this study (Cicerone & Nerger, 1989). The

previous study tested 6 participants: 4 emmetropes and 2 myopes. The mean L/M

cone ratio was 1.97±0.38 of the emmetropes and 2.14±0.21 of the myopes. In our

study, we were planning to recruit three groups: emmetropia, lower myopia and

higher myopia, so there needs to be twice as many myopes as emmetropes.

According to GPower results, to get power value of 0.8, we need 36 emmetropes and

72 myopes. Because of the time limitation of this study and using mfVEP to isolate L

and M cone is a new technique in our laboratory, we recruited 30 participants for this

thesis (10 emmetropes and 20 myopes).

Cone Ratios in Myopia and Emmetropia

52

CHAPTER 3

Cone ratios in myopia and emmetropia

3.1 Introduction How the eye uses visual inputs from the external environment to direct eye growth

remains unclear, although it is well known that the retina plays a crucial role

(Wallman & Winawer, 2004). The retinal processes mediating emmetropization are

thought to integrate visual signals over restricted spatial areas (Wallman et al., 1987)

with both the retinal periphery and fovea being important for controlling eye growth

(Smith et al., 2005; Smith et al., 2007). Thus the signals and activity of the cones

across the retina are speculated to contribute greatly in the development of myopia

(Chen et al., 2006; Rucker & Kruger 2006). In the outer retina, both the functional

response and the structure of cones vary between myopic and emmetropic eyes

(Kawabata & Adachi-Usami, 1997; Liang et al., 1995). For example, the multifocal

electroretinogram (mfERG) amplitudes are reduced and the latencies are delayed in

human myopic eyes (Kawabata & Adachi-Usami, 1997). Photoreceptors are

abnormal in deprivation induced myopic chicks; cone outer segments are damaged

and the cone inner segments are thickened (Liang et al., 1995).

A recent study reported that higher myopic individuals have higher sensitivity to L

cone stimulation (Rucker & Kruger, 2006). In that study of 9 participants, the

participants viewed stimuli with different cone contrast ratios while their

accommodation was measured continuously. Their results indicated that more

myopic individuals showed a higher level of accommodation gain to relatively more

L cone contrast. There are at least two possible explanations for persons with higher


53

myopia having higher sensitivity to L cone stimulation. One interpretation is myopes

have higher L/M cone ratios. The other interpretation is genetic differences in the

spectral sensitivity of the photopigments. An alternative is that the result was found

due to a sampling error due to the small number of participants.

The L/M cone ratios have been studied using various techniques. The proportion of L

and M cones in individuals with normal colour vision varies considerably, ranging

from 0.4 to 13 (Table 1.3). The gene expression analysis reported L/M ratios from

1.3 to 4.9 (Hagstrom et al., 1998; Hagstrom et al., 2000). Direct imaging of the retina

gave L/M ratios from 1.2 to 3.8 (Bowmaker & Dartnall, 1980; Dartnall et al., 1983;

Roorda & Willians, 1988). Psychophysical studies suggested L/M ratios from 1.5 to

2.4 (Otake & Cicerone, 2000; Pokorny & Smith, 1987; Cicerone & Nerger, 1989;

Nerger & Cicerone, 1992), and electrophysiology techniques gave estimated L/M

ratios from 0.4 to 13 (Brainard et al., 2000; Usui et al., 1998; Carroll et al., 2000;

Carroll et al., 2002; Kremers et al., 1999; Langrova et al., 2007; Albrecht et al., 2002;

Hood et al., 2002).

There is little knowledge about L/M cone ratios in myopia and emmetropia. Only

three papers regarding L and M cone ratio mentioned the refraction status of

participants (Cicerone & Nerger, 1989; Nerger & Cicerone, 1992; Otake & Cicerone,

2000) (Table 1.4). From the first paper, the L/M ratios of one emmetrope and one

myope were 1.6 and 2.0. From the second paper, the L/M ratios of four emmetropes

were 2.1, 2.0, 1.5, 2.4, respectively; the L/M ratios of two mild myopes were 2.3 and

2.0. From the third paper, the L/M ratio of one emmetrope was 1.8; the L/M ratios of

two mild myopes were 2.2 and 1.5. These three studies showed similar L/M cone

ratios between myopia and emmetropia, however the sample sizes are small.

The present study investigated the L/M cone ratios in the central retina between

myopic and emmetropic normal trichromats using mfVEP. Although it is suggested

that the L/M ratios may be an important determinant of myopia progression (Rucker

& Kruger, 2006), the fact that individuals with red-green colour vision deficiency are

not all myopic (Qian et al., 2009), leads to the prediction that the L/M cone ratio will


54

vary in individuals but not show a regular difference simply due to refractive error.

3.2 Participants Data were collected on 30 young, colour normal adults aged 22 to 33 years,

including 10 emmetropic (spherical equivalent +1.00D to -0.25D), 10 lower myopic

(spherical equivalent -1.00D to -3.00D) and 10 higher myopic (spherical equivalent

-3.50D to -7.25D) individuals. To establish the reliability of the two stimulus light

conditions for isolating only one type of cone, two protanopes and one deuteranope

were examined. The protanopes and deuteranope were all emmetropic and aged 24,

30 and 36, respectively. The three dichromatic participants had normal

ophthalmological findings with visual acuities of 6/6. The colour vision defect of

these participants was confirmed using the Nagel Anomaloscope. One protanope

showed unreliable responses for the black-white modulation and thus did not proceed

with the rest of the testing. Email was used for recruiting initial participants and also

a snowballing method was used to recruit later participants within the University

community.

All experiments were conducted with human research ethics approval in accordance

with the National Statement on Ethical Conduct in Human Research published by the

National Health and Medical Research Council of Australia. Informed written

consent was obtained from all participants after the purpose and the procedures of

the study were explained. The QUT human research ethics approval number was

1100001318.

To determine suitability of potential participants, a screening eye examination was

conducted, including refraction (autorefractor), colour vision assessment (Ishihara)

and eye health assessment (slit lamp biomicrosopy and ophthalmoscopy). Corrected

visual acuity of 6/6 and normal trichromatic colour vision were required and were

tested by the researcher. Individuals with retinal pathology, abnormal ocular media,

strabismus, glaucoma and photosensitive epilepsy were not eligible to take part in

this study. Based on these criteria, one screened individual was excluded because of


55

amblyopia.

3.3 Procedure

3.3.1 The mfVEP visual stimuli

We used a CRT monitor with resolution of 1152*864 pixels to present the visual

stimuli and used the Visual Evoked Response Imaging System (VERIS 5.1，

Electro-Diagnostic Imaging Inc., San Mateo, CA, USA) to perform mfVEP

stimulation and data collection. Figure 3.1 shows the standard stimulus of the mfVEP.

The stimulus was a dartboard array consisting of 60 sectors and each sector was a

checkerboard pattern consisting of 16 checks. The stimuli were driven at a frame rate

of 75 Hz on the CRT monitor and subtended 30° horizontally and 30° vertically. The

sizes of the elements increased as they extended peripherally in order to give

approximately equal contrast response. Each of the elements was illuminated

following a pseudorandom m-sequence (215-1) to make the mean luminance of the

screen constant over time (Hood et al., 2002). The rod contribution to the mfVEP

was minimised by the bright rapid flicker and keeping the room lights on

(approximately 100 cd/m2).


56

FIGURE 3.1 The standard stimulus of the mfVEP in this study. The black and white elements

were alternated following the 215-1 m-sequence to stimulate the eye. This is a photographic

representation of the stimuli.

The method of silent substitution involves changing the output of three primary

lights, with psychically different spectral distributions, to alter the activity of one

cone class without changing the excitation of the unmodulated cone photoreceptor

classes. Each stimulus element was alternated between two lights selected to isolate

activity only in L-cone or only in M-cone. Figure 3.2 and 3.3 show the stimuli for the

L cone modulation and the M cone modulation applied in this study.


57

Figure 3.2 The stimuli of the L cone modulation. The RGB values of the “red” light are R243, G0,

B0. The RGB values of the “blue” light are R0, G122, B176. The mean luminance was 28.8

cd/m2. This is a photographic representation of the stimuli.


58

Figure 3.3 The stimuli of the M cone modulation. The RGB values of the “green” light are R0,

G177, B0. The RGB values of the “pink” light are R243, G0, B176. The mean luminance is 44.0

cd/m2. This is a photographic representation of the stimuli.

3.3.2 The mfVEP recording

One channel with three gold cup electrodes was used for detecting mfVEP signals.

The ground electrode, the reference electrode and the active electrode were placed on

the forehead, the inion and 4 cm above the inion respectively (see Figure 3.4) (Hood

et al., 2000). To reduce the resistance, the skin and the scalp where the electrodes

placed were cleaned with abrasive skin preparation gel and the electrodes were filled

with electrode cream. The impedance across the two electrodes on the skull was less

than 5 kOhms measured by a GRASS Electrode Impedance Meter (W.WARWICK,

RI U.S.A, F-EZM5).


59

FIGURE 3.4 The placement of the electrodes in this study. In the left picture, the upper electrode

is active electrode which is placed 4cm above the inion and the lower electrode is reference

electrode which is placed on the inion. In the right picture, the ground electrode is placed on the

forehead.

After 15 minutes adaptation to the room illumination, participants were asked to sit

37 cm in front of the monitor. Fixation of a black cross in the centre of the stimulus

display was used for participants. All the participants had natural undilated pupils

(average diameter 5mm) and their left eyes were occluded (see Figure 3.5). The

participants were provided with spherical as well as cylindrical correction by means

of the patients’ eye glasses. During the experiments, the signal level was checked via

the Apple Mac control computer.

ACTIVE ELECTRODE

REFERENCE ELECTRODE

GROUND ELECTRODE


60

FIGURE 3.5 During the experiment, the participant sits 37 cm in front of the monitor with

natural pupil and the left eye is occluded. The electrodes were placed on the scalp to detect the

mfVEP signals. The mfVEP signals were transported to the computer.

3.3.3 Dichromat pilot testing

In the dichromat experiments, the length of the m-sequence was 213-1. Each run

lasted 1 minute and 49 seconds and was divided into 8 overlapping segments, each

segment lasted 13.65 seconds. Seven runs with different colour combinations were

recorded and each run was repeated, so there were fourteen runs in total. Table 3.1

shows the colour combinations of each run. Because big change of the modulations

in this study is not easy to find the lowest amplitude of mfVEP signals, we adjusted

the change of the modulations within 10%.


61

TABLE 3.1 The colour combinations of dichromat experiments

Run Protanope Deuteranope

1 Black / White Black / White

2 L modulation (100%R / 15.7%G+1.6%B) L modulation (100%R / 15.7%G+1.6%B)

3 M modulation (43.1%G / 100%R+1.6%B) M modulation (43.1%G / 100%R+1.6%B)

4 +2.5% L modulation (100%R / 18.2%G+1.6%B) +2.5% M modulation (45.6%G / 100%R+1.6%B)

5 +5% L modulation (100%R / 20.7%G+1.6%B) +5% M modulation (48.1%G / 100%R+1.6%B)

6 -2.5% L modulation (100%R / 13.2%G+1.6%B) -2.5% M modulation (40.6%G / 100%R+1.6%B)

7 -5% L modulation (100%R / 10.7%G+1.6%B) -5% M modulation (38.1%G / 100%R+1.6%B)

The percentage numbers stand for the energy percent of phosphor. For example, to

obtain 100%R, the bit number should be set at 255. The bit number of each

modulation can be chosen from Appendix I, II and III. Table 3.2 shows the

corresponding bit number of each modulation.


62

TABLE 3.2 The corresponding bit number of each modulation

Light 1 (R, G, B) Light 2 (R, G, B)

L modulation 243, 0, 0 0, 122, 176

+2.5% L modulation 243, 0, 0 0, 132, 176 +5% L modulation 243, 0, 0 0, 137, 176

-2.5% L modulation 243, 0, 0 0, 115, 176 -5% L modulation 243, 0, 0 0, 111, 176

M modulation 0, 177, 0 243, 0, 176 +2.5% M modulation 0, 181, 0 243, 0, 176

+5% M modulation 0, 185, 0 243, 0, 176 -2.5% M modulation 0, 174, 0 243, 0, 176

-5% M modulation 0, 169, 0 243, 0, 176

3.3.4 Trichromat experiments

In the trichromat experiments, the m-sequence was 215-1. The recording of L cone

modulation or M cone modulation lasts 7 minutes and 17 seconds. To assist

participants in maintaining good fixation, the recordings were divided into 16

overlapping sessions, each lasting 27.31 seconds. The participants were encouraged

to keep fixing at the cross in the centre of the stimulus during the recording. After

one L cone modulation and one M cone modulation had been finished, each


63

modulation was repeated one more time, so there are two runs of the L cone

modulation and two runs of the M cone modulation.

3.3.5 Exporting mfVEP data

To increase the signal to noise ratio, the mfVEP outputs were grouped and averaged

(Hood et al., 2000). The data of mfVEP signals of each participant were exported

into 16 groups via a USB to Excel spreadsheets (Figure 3.6). The first slice of the

second-order kernel was extracted. In an excel spreadsheet, the mfVEP signals were

summed and averaged to one curve for each participant. For example, each

trichromat has four mfVEP recordings: L modulation I, L modulation II, M

modulation I and M modulation II. To get the mfVEP signals within 3°-13° of L

cones, averaged the groups of 3, 4, 5, 11, 12, 13 of L modulation I and L modulation

II, respectively, and then averaged the summed L modulation I and L modulation II

to one plot. Similarly, averaged the groups of 6, 7, 8, 14, 15, 16 to obtain mfVEP

signals within 13°-30° of L cones. A similar process was done for mfVEP signals of

M cones within 3°-13° and 13°-30°.

13°

30°

3°


64

FIGURE 3.6 The 16 groups of the mfVEP signals exported from mfVEP. The corresponding 3°

retina includes groups 1, 2, 9 and 10. The corresponding 3°- 13° retina includes groups 3-5 and

11-13. The corresponding 13°- 30° retina includes groups 6-8 and 14-16.

3.4 Results

3.4.1 The mfVEP responses of dichromats

Dichromats have only two types of functional cones. Protanopes have M and S cones

and deuteranopes have L and S cones. The L modulation signal should produce the

lowest amplitude of mfVEP response in the protanopes (Figure 3.7, 3.8 and 3.9),

whereas the M modulation signal should be least for the deuteranopes (Figure 3.10,

3.11 and 3.12).


65

FIGURE 3.7 The summed and averaged mfVEP signals from one protanope. Each curve is the

average of two 1 minute and 49 seconds runs of mfVEP recordings. The black curve is from

standard mfVEP stimuli to make sure this protanope has normal mfVEP responses. The

amplitude of the red curve is much smaller than the green curve as protanopes do not have

functional L cones.


66

FIGURE 3.8 The summed and averaged mfVEP signals from one protanope. Each curve is the

average of two 1 minute and 49 seconds runs of mfVEP recordings. To test if the set of L

modulation gives the minimum mfVEP signal from a protanope, we adjusted the set of L

modulation and compared the amplitude of each set. All the curves are less than the M

modulation in Figure 3.7, because the stimuli used to generate these curves do not fully excite M

cones.


67

FIGURE 3.9 The amplitude of adjusted modulations from one protanope. The amplitude of

black/white, M modulation, L modulation, +2.5% L modulation, +5% L modulation, -2.5% L

modulation and -5% L modulation are 1.33E-6 V, 1.30E-6 V, 6.05E-7 V, 9.02E-7 V, 1.03E-6 V,

1.22E-6 V, 8.33E-7 V respectively. The smallest amplitude is the protanope mfVEP signal to the

L modulation.


68

FIGURE 3.10 The summed and averaged mfVEP signals from one deuteranope. Each curve is

the average of two same 1 minute and 49 seconds runs of mfVEP recordings. The black curve is

from standard mfVEP stimuli to make sure this deuteranope participant has normal mfVEP

responses. The amplitude of the green curve is much smaller than the red curve as deuteranopes

do not have functional M cones.


69

FIGURE 3.11 The summed and averaged mfVEP signals from one deuteranope. Each curve is

the average of two 1 minute and 49 seconds runs of mfVEP recordings. To prove the set of M

modulation gives the minimum mfVEP signal from a deuteranope, we adjusted the set of M

modulation and compared the amplitude of each set. All the curves are less than the L modulation

in Figure 3.10, because the stimuli used to generate these curves do not fully excited L cones.


70

FIGURE 3.12 The amplitude of adjusted modulations from one deuteranope. The amplitude of

black/white, L modulation, M modulation, +2.5% M modulation, +5% M modulation, -2.5% M

modulation and -5% M modulation are 1.18E-6 V, 1.16E-6 V, 3.68E-7 V, 4.87E-7 V, 5.57E-7 V,

5.47E-7 V, 5.57E-7 V respectively. The smallest amplitude is the deuteranope mfVEP signal to

the M modulation.

Theoretically, as protanopes do not have functional L cones, the mfVEP signal of L

modulation should be zero and as deuteranopes do not have functional M cones, the

mfVEP signal of M modulation should be zero. However, the result of the protanope

shows a small mfVEP signal under L modulation in Figure 3.7 and the result of

deuteranope shows a small mfVEP signal under M modulation in Figure 3.10. The

same cone isolating methodology has been used in both mfERG and mfVEP

experiments (Albrecht et al., 2002; Hood et al., 2002). The mfERG study obtained

zero signals of L cone modulation for protanopes and zero signals of M cone

modulation for deuteranopes, however the mfVEP study did not obtain zero signals

using the same cone isolation stimuli. It is not known why a small rather than zero

signals may have been measured, but it is thought that the dartboard visual stimulus


71

of the mfVEP is more complex than the hexagonal visual stimulus array of the

mfERG and the smaller stimulus areas of the dartboard array might introduce some

other signals.

Another possibility is that the post-receptoral pathways exchange information

between each other. The electrical signal travelling through a nerve due to M cone

activation in a protanope, for instance, might induce a signal in a neighbouring

pathway originally associated with L cones. Therefore, detecting the stimulation at

the retinal level will yield a zero signal while detecting the signal at a later stage will

show a weak signal for the L cone, around the same time of the M cone signal, but

with a much lower amplitude. The phenomenon described above is referred to as

“nerve cross talk”, and was reported earlier by Gardner et al (Gardner, 1966). This is

one possible explanation for the non-zero signal detected by the mfVEP without L

cone stimulation. Moreover, there might be small contribution from the S cones and

rods (see section 4.5).

Figure 3.9 and 3.12 compared the amplitudes of adjusted L modulations and M

modulations. In the results of the protanope participant, the black/white stimulus has

the highest amplitude and then the M modulation. The lowest amplitude is the L

modulation, but the other four adjusted L modulations did not give an expected

amplitude order. For example, the amplitudes of -5% L modulation should be higher

than the -2.5% L modulation, however the results are opposite. The results of the

deuteranope participant revealed a similar problem. The unexpected relationship of

the amplitudes between the adjusted modulations might because of “nerve cross talk”.

The visual pathway system is a very complex system. Visual nerves influence

between each other (Gardner, 1966). Another reason could be that the participants

fixated to the stimuli differently. The mfVEP responses are sensitive to the change of

gaze position of the participants (Menz & Sutter, 2004).


72

3.4.2 The mfVEP responses of all the trichromats

The mfVEP responses of the central 3°-13° retina (Figure 3.13) and peripheral 13°-30°

retinal ring (Figure 3.14) of all trichromat participants were summed and averaged.

The data of foveal 3° retina were excluded because there is a gain adjustment in the

neural pathway in the foveal retina, resulting the L/M cone ratio to 1 (Hood et al.,

2002). Because of poor mfVEP signals (no typical N1-P1 amplitudes), the 3°-13°

data of participants SS, SV, NC and RJY and the 13°-30° data of participants SS, MC,

NC and IF were excluded (indicated by circles around the mfVEP data shown in

Figure 3.13 and 3.14).


73

CR

JYZ

DJ

PT

YH

YF

ZNY

SS

AW

SV

QM

MLC

MC

AM

CD

NC

WCC

FS

JG

CMJ

SPP

XZW

HPP

RJY

YSB

IF

WJJ

HY

MGL

ZXF

200 ms 1 µV


74

FIGURE 3.13 The summed and averaged mfVEP signals in the central 3°-13° retina of all

trichromat participants. The red curve is the average of two same L modulations and the green

curve is the average of two same M modulations. The ellipses represent poor mfVEP responses

that were deleted. Data of participants SS, SV, NC and RJY were excluded.


75

CR

JYZ

DJ

PT

YH

YF

ZNY

SS

AW

SV

QM

MLC

MC

AM

CD

NC

WCC

FS

JG

CMJ

SPP

XZW

HPP

RJY

YSB

IF

WJJ

HY

MGL

ZXF

200 ms 1 µV


76

FIGURE 3.14 The summed and averaged mfVEP signals in the 13°-30° retina of all trichromat

participants. The red curve is the average of two same L modulations and the green curve is the

average of two same M modulations. The ellipses represent poor mfVEP responses that were

deleted. Data of participants SS, MC, NC and IF were excluded.


77

3.4.3 The L/M ratios of trichromats

Table 3.3 lists the information of normal trichromats and their L/M ratios.

TABLE 3.3 The L/M ratios of all normal trichromat participants.

Participant Gender Age Refraction(D) L/M ratio 3°-13° L/M ratio 13°-30°

CR M 24 +1.00 1.09 1.87

JYZ F 27 +0.75 1.68 1.63

DJ M 28 +0.50 1.13 1.43

PT F 22 +0.50 1.50 1.19

YH M 24 +0.25 1.09 1.49

YF M 32 +0.25 1.06 1.91

ZNY F 31 0.00 1.17 1.18

SS F 33 0.00 — —

AW F 23 0.00 0.87 1.29

SV M 27 -0.25 — 1.36

QM M 23 -1.00 1.46 0.96

MLC F 28 -1.00 1.21 1.23

MC M 22 -1.25 1.29 —

AM M 29 -1.50 1.32 1.55

CD M 33 -2.50 1.71 1.77

NC M 30 -2.50 — —

WCC F 25 -2.75 1.19 1.66

FS F 27 -3.00 0.86 1.20

JG F 23 -3.00 1.43 1.28

CMJ F 28 -3.00 1.05 1.33

SPP F 23 -3.50 1.23 1.48

XZW M 24 -4.00 1.80 1.44

HPP F 28 -4.00 0.77 1.00

RJY M 29 -4.00 ― 1.10

YSB M 31 -4.00 1.05 1.75

IF M 24 -4.00 1.45 —

WJJ F 26 -4.50 0.56 1.12

HY M 28 -4.50 0.73 0.94

MGL M 33 -7.00 1.37 1.18

ZXF F 25 -7.25 1.14 1.08


78

3.4.4 Data analysis between emmetropic, low myopic and high myopic groups

The One-way ANOVA analysis was conducted. When applying one-way analysis of

variance, three following assumptions should be satisfied: i) the populations from

which the samples were obtained should be normally or approximately normally

distributed; ii) the samples must be independent; iii) the variances of the population

must be equal. In this study, we recruited 30 participants and tested their own

LWS/MWS ratios, so the observations in this study were independent. The results of

the homogeneity of variances test revealed that there were no significant differences,

and the results showed that the normal distribution of population from which the

samples were recruited was not violated.

Within the central 3°-13° of retina, the L/M cone ratios of the emmetropic, low

myopic and high myopic groups were 1.20 ± 0.26, 1.28 ± 0.25 and 1.12 ± 0.39,

respectively. There were no significant differences in the value of L/M cone ratio

amongst the three groups (p=0.565).

Within the 13°-30° retinal ring, the L/M cone ratios of the emmetropic, low myopic

and high myopic groups were 1.49 ± 0.27, 1.37 ± 0.27 and 1.23 ± 0.27, respectively.

There were no significant differences in the value of L/M cone ratio amongst the

three groups (p=0.161).

3.4.5 Data analysis between emmetropic and myopic groups

All myopes were being grouped together and the independent-samples t-test analysis

was conducted. Three assumptions should be met for conducting the

independent-samples t-test: i) the populations from which the samples were obtained

should be normally or approximately normally distributed; ii) the samples must be

independent; iii) the variances of the population must be equal. This study tested the

participants’ own L/M ratio, so the observations in this study were independent, and

the homogeneity of variances test showed that here were no significant differences

and the normal distribution of population from which the samples were recruited was

not violated.


79

Within the central 3°-13° of retina, the L/M cone ratios of the emmetropic and

myopic group were 1.20 ± 0.26 and 1.20 ± 0.33, respectively. The L/M ratios of the

two groups were not different in the central ring (p=0.986).

Within the 13°-30° retinal ring, the L/M cone ratios of the emmetropic and myopic

group were 1.48 ± 0.27 and 1.30 ± 0.27, respectively. The L/M ratios of the two

groups were not different in the mid-peripheral ring (p=0.108).

3.4.6 Data analysis between central and peripheral retina

Paired-samples t-test was conducted between central 3°-13° and peripheral 13°-30°

of L/M cone ratios in 24 trichromat participants (SS, SV, NC, RJY, MC and IF were

excluded). There was significant statistic difference of L/M cone ratios between the

central and peripheral retina (p=0.017). The L/M cone ratios of the central retina

were 1.19±0.31. The L/M cone ratios of the peripheral retina were 1.37±0.29.

3.4.7 Post hoc effect size analysis

Although there was no statistical difference in the L/M ratios between myopes and

emmetropes, there was a trend toward significance for the most peripheral retinal

ring tested. Thus a post hoc power analysis was conducted on the peripheral data to

predict effect size for L/M ratio studies using mfVEP.

For one-way ANOVA, according to the data in the peripheral ring of current study,

the power value was 0.38 using GPower (3.1.5) software, and in order to get a power

value of 0.8, 66 participants in total are required.

For unpaired t-tests, according to the data in the peripheral ring of current study, the

power value was 0.34 using GPower (3.1.5) software, and in order to get a power

value of 0.8, 82 participants in total are required.

Discussion

79

CHAPTER 4

Discussion The present study estimated the L/M cone ratios of the central and mid-peripheral

retina using the mfVEP in 30 participants with normal colour vision. Statistical

analysis indicated that there were no significant differences in the L/M cone ratio

between the emmetropic, lower myopic and higher myopic groups for both the

central 3°-13° retina (p=0.565) and 13°-30° ring (p=0.161). There were also no

significant differences in L/M cone ratios between the emmetropic group and the

pooled data of all myopes, i.e to form one myopic group. The L/M cone ratios in

normal trichromat participants, comparisons between myopes and emmetropes, as

well as the reliability of the cone isolation stimuli and the limitations of the L/M

cone ratio estimates will be discussed.

4.1 Cone ratios in trichromats

In all normal trichromatic participants, the total L/M ratio ranged from 0.56 to 1.80

within the central 3°-13° and from 0.94 to 1.91 within the 13°-30° ring. A previous

study used mfVEP to estimate the L/M ratios in 6 colour normal trichromats (Hood

et al., 2002); the L/M ratio ranged from 1.1 to 1.7 within the central ring and 5.8° to

22.2° mid-peripheral ring. The range of L/M ratios reported here are thus similar to

the range reported in that paper; particularly given the small differences in visual

field area and the larger sample size (n=30) in this study.

Some of the factors that may impact on mfVEP responses have been investigated,

with Winn et al. (2005) concluding that mfVEP responses are affected by simulated

refractive errors, cataracts and fixations errors; these were not issues for the young,

Discussion

80

spectacle corrected participants of this study. The poor mfVEP responses were from

those participants who easily go to sleep during the experiments and were excluded

from the data. The test re-test reliability of the mfVEP is stated to be good, with an

r=0.85 for amplitude measures made on two separate days (Chen et al., 2003). Here

the agreement between the two test runs was r=0.95 for the L modulation and r=0.95

for the M modulation.

Other techniques have also been used to estimate L/M cone ratios. L/M cone ratios

determined using gene expression range from 1.3 to 4.9 in the central 40° of retina.

Direct imaging from the foveal retina gave L/M ratios ranging from 1.15 to 3.79.

Several psychophysical studies have been carried out and reported L/M ratios range

from 1.1 to 3.4 in the central 28° of retina. Electrophysiology has been widely used

to estimate L/M ratios, which range from 0.4 to 13 across the 124° of the retina; the

extreme values were rare, eighty percent of the L/M ratios were within the range of

1.0-4.0. These studies report a larger range of L/M cone ratios than our study, and

this probably reflects the difference in measured retinal areas and the principals of

the technique employed. For example, electrophysiological study which can measure

retinal area up to 124° gives L/M cone ratio from 0.4 to 13.

4.2 Myopes vs emmetropes

There was no statistical difference between the L/M ratio of the emmetropic and

myopic groups. Given the fact that not all protanopes are emmetropic and not all

deuteranopes are myopic, we predicted the L/M cone ratio was not a crucial factor in

the development of myopia. The finding of no difference of L/M cone ratios between

myopic and emmetropic groups is consistent with this prediction.

Some studies have suggested the L and M cone activities are related to myopic

refractive error. One small study has been carried out to determine the effect on

myopia progression of balancing the L and M cone activities in children (Neitz &

Neitz, 2011). This study indicated special eye glasses which equalize the L and M

cone activities reduced myopia progression to 10 times slower than that observed

Discussion

81

with normal eyeglasses. Another study has demonstrated that myopes are more

sensitive to the long-wavelength component (Rucker & Kruger, 2006). They

explained this may be due to a shift in cone pigment sensitivity towards

long-wavelength in myopia or relatively more L cones number than M cones in

myopia.

If the higher sensitivity of L cones related to myopia, it could be assumed that

protanopes without any L cones should not develop myopia. In contrast,

deuteranopes without any M cones should be myopic. One paper investigated the

prevalence of myopia in 309 red-green colour deficiency students (Qian et al., 2009).

They found that 43.7% protanopes and 47.3% deuteranopes are myopic, i.e the

prevalence of myopia was the same. This finding seems to challenge the above

assumption. While in the colour normal 927 students, myopia was present in 65.8%.

The difference of the prevalence in colour deficiency and colour normal group was

significant (random-effects logistic test: p<0.001). Therefore, the result that

red-green colour deficiency group had a lower prevalence of myopia cannot be

simply explained by L/M cone ratios. The L and M chromatic opponent has reduced

function in the red-green colour deficiency group, but the S chromatic opponent has

not been significantly affected.

In the 13°-30° diameter ring, the trend was for the L/M cone ratio to be lower in the

myopes; this is opposite to the predicted direction of the difference of a greater L/M

cone ratio in myopia. A post hoc power analysis of the peripheral ring data suggests

that with a power of 0.8, 27 participants in each group would be required to detect a

significantly lower cone ratio in myopes than in emmetropes in the peripheral ring. A

difference in this direction (i.e. lower L/M ratio in myopia), while not related to

myopia development per se, may occur as a consequence of myopia. Alternately a

recent study suggests that myopes may have lower L/M cone ratios; although this

was based on data of only 6 participants (Yamauchi et al., 2013). The lower L/M

ratio in myopes in Yamauchi’s study was consistent with the results in our study.

The peripheral eye shape could be considered as a reason that influences myopia

Discussion

82

development in the colour deficiency students. Most of the cones only take over a

small area of the fovea retina. The much bigger peripheral area of the retina may play

an important role in myopic progression. It has been reported that emmetropes and

myopes have different pattern of peripheral refractive errors (Charman, 2005; Mutti

et al., 2007; Seidemann et al., 2002; Stone & Flitcroft, 2004).

4.3 Central retina vs peripheral retina

There was a significant difference between the L/M cone ratio for the central 3°-13°

and peripheral 13°-30° rings. The L/M cone ratios of the central retina were lower

than that of the peripheral retina. This result is consistent with other L/M cone ratio

studies using different methods. In the mRNA analysis, the L/M cone ratios of the 20°

and 40° retina were 1.5 and 3.0 respectively (Hagstrom et al., 1998). The multifocal

pattern electroretinogram technique estimates of the L/M cone ratios for the 8° and

12.8°-26° retina areas were 1.00-2.78 and 1.29-2.78 respectively (Langrová et al.,

2007). The mfERG measurement suggested the L/M cone ratios were 1.4 for the

central 5° retina and 2.3 of the 40° retina (Albrecht et al., 2002).

This difference in estimated L/M ratio across the retina suggest the number of L

cones increases with retina excentricity or the number of M cones decreases with

retina excentricity. Direct images of trichromatic cone mosaic in human can address

this question. However, studies with this technique only measured the cone mosaic in

the foveal retina (Roorda, & Williams, 1988).

4.4 Validity of the cone isolating stimuli

The Smith and Pokony 10° fundamentals were used to calculate L and M isolating

stimuli in this study. Other fundamentals are also available for the calculation, e.g.

Smith and Pokony 2° fundamentals. As this study investigated the L/M cone ratios

for 30° retina, we chose the fundamentals calculated for the larger retina area. In fact,

using different fundamentals is stated to give only minor changes in the calculated

parameters of cone isolation stimuli (Kremers et al., 1999). This study did not correct

Discussion

83

for individual differences in pre-receptoral filtering by the lens or macular pigment,

which will affect cone isolation. The effect of lens attenuation however, would be

small due to the younger age range of the sample (22-33 years old).

The luminance output of CRT monitors change with the temperature and the

luminance output remain stable after they have been turned on for at least 40

minutes (Metha et al., 1993). All the calibrations and experiments described in this

thesis were conducted after the monitor was allowed to warm up for 1 hour; this

avoided the influence of luminance changes on cone isolating stimuli.

The cone isolating stimuli were also tested on one protanope and one deuteranope.

To minimize any unwanted signals, the cone isolating settings were adjusted slightly,

and we chose the best parameters (see section 3.4.1).

4.5 Limitation of the mfVEP study of L/M cone ratios

It might be possible that there are some small S cone and rod signals. We used a

three primaries CRT to display the mfVEP stimuli, but to isolate one of the

photoreceptor classes, four primaries are required (Zele et al., 2012). In this study,

the L or M cone was isolated from other types of cones by the silent substitution

technique. The activity of the rods was minimised by prior light adaption and a fast

flickering stimuli. The 75 Hz frame rate of the CRT monitor and the mean luminance

of the L and M cone modulation were suggested not bright enough to separately

investigate L or M cone responses (Kremers et al., 2009; Zele & Vingrys, 2005). The

average pupil size was 5 mm during the mfVEP measurements. The retinal

illuminance of L modulation and M modulation were approximately 565.2 Td and

863.5 Td, respectively, during experiments. It has been suggested that if the retinal

illuminance is below 1000 Td (Zele et al., 2008), the rod signals might impact on the

results.

The PC and MC pathways are known to process information from both L and M

cones (Lee, 1996). In the foveal 1° retina, mfVEP responses mainly come from the PC

Discussion

84

pathway, because 95% of the ganglion cells in the fovea are midget ganglion cells

which subject to the PC pathway (Dacey, 1993). It is suggested that a gain

adjustment resulting 1:1 L/M ratio in the PC pathway has taken place after the

mfERG but before the mfVEP is generated in the central fovea (Hood et al., 2002).

Outside the foveal retina, mfVEP combine signals mainly from both PC and MC

pathways, because the midget ganglion cells projecting PC pathway make up around

45% and the parasol ganglion cells projecting MC pathway contribute 20% of the

total ganglion cells in the peripheral retina (Dacey & Petersen, 1992). In this study,

the MC pathway is already saturated at high contrast (Baseler & Sutter, 1997), so the

combination of PC and MC pathways in mfVEP response is not linear to the

numerosity of cones. Moreover, the positive and negative peaks of mfVEP signals

from the PC and MC pathways can cancel out each other (Baseler & Sutter, 1997).

According to the results of this study, the sample size might be not enough to detect

real L/M cone ratio differences in the peripheral retina of myopic and emmetropic

groups. Although both one-way ANOVA and independent-samples t-test analysis

indicated similar L/M cone ratios in the central retina, the variation of the L/M cone

ratios in the peripheral retina were greater between individuals. The P value of

one-way ANOVA and independent-samples t-test analysis in the peripheral retina

were 0.161 and 0.108. Power calculations suggest that increasing the total participant

numbers to 27 in each group would be required to prove whether the lack of

difference is due to insufficient power or is actually a real difference. In any case, the

effect size is likely to be very small if the effect were real.

The practical limitations of recording mfVEP are the placement of electrodes and the

fixation of participants during experiments. A proper and clean electrodes placement

can reduce the signal to noise ratio of the mfVEP responses. Before starting

measurements, the scalp-electrode impedances were checked and measurements only

proceeded when these were below 5 kOhms. This study used one channel electrodes

placement, it has been suggested that additional electrodes and channels can improve

the mfVEP signals and reduce the signal to noise ratio (Hood et al., 2002). The

fixation of participants during mfVEP tests was checked visually by the examiner.

Discussion

85

An objective method using a camera to monitor the fixation can more accurately

monitor fixation; minor changes of fixation can produce large variations of the

amplitude of mfVEP signals (Menz & Sutter, 2004).

4.6 Future directions

A relative large number of participants were tested in this study, compared to

previous research, using mfVEP to determine L/M ratios. No statistical difference

has been detected in this study; according to the post hoc power analysis, 27

participants were required of each group to find such differences. This study with 10

participants of each group provided basic data for the future L/M ratios studies using

mfVEP. In the future study, more participants will be recruited to meet the

requirement of powers of at least 0.8.

This study recruited about 70% Asian participants. It is known that the prevalence of

myopia of Asia countries is the highest over the world, so the high proportion of

Asian participants in this study could bias the results to this group. Recruiting

different ethnic groups with balanced number of participants could answer the

question whether the L/M cone ratio only impacts myopia for some racial groups.

However, the results from the mfVEP indicate that there are no significant individual

differences in cone ratios between persons with emmetropia or myopia and thus

differences in the suggested experiments unlikely. The future study can match one

Asian participant to one Western participant to balance the ethnic bias. Alternatively,

only one racial group can be considered. There has been a study suggesting that

myopes may have lower L/M cone ratios in Japanese, but it only had 6 participants

(Yamauchi et al., 2013).

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86

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Appendix I

96

Appendix I

Original luminance output (cd/m2) of red light from the CRT monitor

1 0.321 38 0.321 75 2.613 112 5.272 149 12.32 186 20.22 223 30.77

2 0.321 39 0.321 76 2.613 113 6.875 150 13.04 187 20.44 224 30.77

3 0.321 40 0.321 77 2.613 114 7.282 151 13.04 188 20.44 225 30.77

4 0.321 41 0.321 78 2.613 115 8.116 152 13.98 189 21.39 226 32.6

5 0.321 42 0.321 79 2.619 116 8.263 153 13.98 190 21.39 227 33.19

6 0.321 43 0.321 80 3.326 117 8.263 154 14.18 191 22.36 228 33.19

7 0.321 44 0.321 81 3.326 118 8.118 155 13.99 192 23.11 229 33.19

8 0.321 45 0.321 82 3.326 119 8.118 156 14.18 193 23.11 230 32.9

9 0.321 46 0.321 83 3.326 120 8.271 157 14.18 194 23.36 231 32.9

10 0.321 47 0.321 84 3.326 121 8.271 158 14.18 195 23.36 232 33.16

11 0.321 48 0.321 85 3.326 122 8.271 159 14.77 196 23.36 233 34.64

12 0.321 49 0.9377 86 3.326 123 9.455 160 14.77 197 24.12 234 35.25

13 0.321 50 0.9377 87 3.326 124 9.455 161 14.77 198 24.64 235 35.25

14 0.321 51 0.9377 88 3.326 125 9.455 162 14.77 199 24.64 236 35.25

15 0.321 52 0.9435 89 3.326 126 8.261 163 15.79 200 24.11 237 35.25

16 0.321 53 0.9435 90 3.326 127 9.925 164 16.89 201 24.62 238 35.57

17 0.321 54 0.9435 91 3.079 128 9.457 165 16.89 202 24.62 239 36.49

18 0.321 55 0.9435 92 3.328 129 8.251 166 17.31 203 24.86 240 35.23

19 0.321 56 0.9416 93 5.166 130 10.08 167 17.31 204 24.86 241 36.5

20 0.321 57 0.9475 94 3.418 131 9.453 168 17.31 205 25.91 242 36.5

21 0.321 58 0.9475 95 3.418 132 9.927 169 17.97 206 26.44 243 36.77

22 0.321 59 0.9475 96 3.944 133 9.927 170 17.97 207 26.96 244 38

23 0.321 60 0.9475 97 3.944 134 9.927 171 17.97 208 26.96 245 38.03

24 0.321 61 0.9455 98 3.944 135 9.927 172 17.3 209 26.96 246 38.03

25 0.321 62 0.9455 99 5.162 136 10.09 173 18.84 210 26.96 247 38.03

26 0.321 63 0.9455 100 5.162 137 10.09 174 19.06 211 28.06 248 38.03

27 0.321 64 1.656 101 5.162 138 10.09 175 19.06 212 28.06 249 38.03

28 0.321 65 1.666 102 5.162 139 10.09 176 19.06 213 28.06 250 38.03

29 0.321 66 1.666 103 5.162 140 10.57 177 19.06 214 28.3 251 38.03

30 0.321 67 1.666 104 5.162 141 10.57 178 19.06 215 28.3 252 38.03

31 0.321 68 1.607 105 5.272 142 11.62 179 19.98 216 28.57 253 38.05

32 0.321 69 1.607 106 5.272 143 12.15 180 20.22 217 28.57 254 38.05

33 0.321 70 1.66 107 5.272 144 12.15 181 20.22 218 28.57 255 38.05

34 0.321 71 1.66 108 6.879 145 12.32 182 20.22 219 30.5

35 0.321 72 1.66 109 6.879 146 12.32 183 20.22 220 29.36

36 0.321 73 1.66 110 6.879 147 12.32 184 20.44 221 30.51

37 0.321 74 2.613 111 5.272 148 12.32 185 20.22 222 30.51

Appendix I

97

Original luminance output (cd/m2) of green light from the CRT monitor

1 0.3289 38 1.834 75 11.16 112 25.57 149 44.39 186 72.41 223 102.5

2 0.3289 39 1.834 76 11.16 113 26.02 150 46.9 187 72.41 224 104.5

3 0.3289 40 1.94 77 11.16 114 26.69 151 46.9 188 74.71 225 105.5

4 0.3289 41 1.94 78 11.16 115 27.46 152 47.56 189 74.71 226 106.3

5 0.3289 42 1.94 79 12.05 116 27.46 153 48.16 190 74.71 227 106.3

6 0.3289 43 1.94 80 12.05 117 28.42 154 48.16 191 76.23 228 108.2

7 0.3289 44 1.94 81 12.05 118 28.92 155 50.12 192 77.02 229 109

8 0.3289 45 2.68 82 12.99 119 28.92 156 50.12 193 77.81 230 111

9 0.3289 46 4.53 83 13.3 120 28.42 157 50.74 194 78.61 231 111

10 0.3289 47 4.53 84 13.96 121 29.92 158 52.08 195 78.61 232 112

11 0.3289 48 4.53 85 13.96 122 30.43 159 52.08 196 80.2 233 113.9

12 0.3289 49 4.53 86 14.3 123 30.43 160 52.65 197 81 234 113.9

13 0.3289 50 4.53 87 14.62 124 30.99 161 54.17 198 81 235 114.8

14 0.3289 51 4.71 88 14.62 125 32.01 162 54.84 199 82.58 236 115.7

15 0.3289 52 5.669 89 15.66 126 32.49 163 55.55 200 83.4 237 115.7

16 0.3289 53 5.669 90 16.75 127 33.58 164 55.55 201 84.29 238 116.7

17 0.3289 54 5.669 91 17.12 128 33.58 165 55.55 202 85.15 239 118.7

18 0.3289 55 5.669 92 16.75 129 34.11 166 57.65 203 85.15 240 120.6

19 0.3289 56 5.669 93 17.12 130 35.24 167 57.71 204 86.79 241 119.7

20 0.3289 57 5.871 94 17.87 131 35.75 168 58.36 205 86.79 242 121.5

21 0.3289 58 5.871 95 18.64 132 35.75 169 59.82 206 88.41 243 121.5

22 0.3289 59 5.871 96 18.64 133 36.28 170 60.5 207 88.41 244 122.4

23 0.3289 60 6.971 97 19.51 134 37.39 171 60.5 208 90.22 245 124.4

24 0.3289 61 6.971 98 19.12 135 36.28 172 61.91 209 90.97 246 125.4

25 0.3289 62 6.971 99 19.12 136 37.97 173 61.91 210 90.97 247 126.3

26 0.3289 63 6.971 100 19.52 137 38.5 174 62.61 211 91.81 248 126.3

27 1.118 64 8.408 101 19.52 138 39.09 175 64.11 212 93.58 249 129.2

28 1.118 65 6.975 102 21.6 139 40.25 176 64.8 213 93.58 250 128.2

29 1.122 66 8.4 103 21.19 140 40.25 177 65.55 214 95.3 251 129.2

30 1.122 67 8.662 104 21.19 141 40.79 178 66.29 215 95.3 252 132.4

31 1.122 68 9.719 105 21.19 142 42.58 179 67.02 216 95.3 253 132.4

32 1.122 69 9.719 106 21.57 143 42.58 180 67.79 217 97.99 254 133.3

33 1.834 70 9.719 107 23.75 144 42.58 181 69.27 218 98.9 255 134

34 1.834 71 10.55 108 24.21 145 42.58 182 70.06 219 99.76

35 1.834 72 10.55 109 24.21 146 44.39 183 69.29 220 100.7

36 1.834 73 11.16 110 24.21 147 45.01 184 70.84 221 100.7

37 1.834 74 10.55 111 24.65 148 45.01 185 71.57 222 101.6

Appendix I

98

Original luminance output (cd/m2) of blue light from the CRT monitor

1 0.323 38 0.323 75 0.323 112 1.259 149 4.902 186 6.902 223 11.15

2 0.323 39 0.323 76 0.323 113 1.259 150 4.902 187 6.902 224 11.15

3 0.323 40 0.323 77 0.323 114 1.259 151 4.902 188 6.902 225 11.15

4 0.323 41 0.323 78 0.323 115 2.018 152 4.902 189 6.902 226 11.25

5 0.323 42 0.323 79 0.323 116 2.063 153 4.902 190 6.902 227 11.16

6 0.323 43 0.323 80 0.323 117 2.063 154 4.902 191 7.828 228 11.16

7 0.323 44 0.323 81 0.323 118 2.063 155 4.902 192 7.828 229 11.16

8 0.323 45 0.323 82 0.323 119 2.063 156 4.902 193 7.828 230 11.16

9 0.323 46 0.323 83 0.323 120 2.063 157 4.902 194 7.828 231 11.16

10 0.323 47 0.323 84 0.323 121 2.063 158 4.902 195 7.828 232 11.26

11 0.323 48 0.323 85 0.323 122 2.063 159 4.962 196 8.923 233 11.26

12 0.323 49 0.323 86 0.323 123 2.063 160 4.962 197 8.923 234 12.59

13 0.323 50 0.323 87 0.323 124 2.063 161 4.962 198 8.923 235 12.59

14 0.323 51 0.323 88 0.323 125 2.063 162 4.962 199 7.836 236 12.59

15 0.323 52 0.323 89 0.323 126 2.063 163 4.962 200 8.924 237 12.59

16 0.323 53 0.323 90 0.323 127 2.063 164 4.962 201 8.924 238 12.59

17 0.323 54 0.323 91 0.323 128 2.063 165 4.962 202 8.924 239 12.59

18 0.323 55 0.323 92 2.018 129 2.063 166 4.968 203 9.51 240 12.59

19 0.323 56 0.323 93 1.257 130 2.063 167 4.968 204 8.928 241 14.22

20 0.323 57 0.323 94 0.323 131 2.063 168 6.812 205 9.394 242 14.22

21 0.323 58 0.323 95 0.323 132 2.063 169 4.974 206 9.498 243 14.22

22 0.323 59 0.323 96 0.323 133 2.063 170 6.065 207 10.4 244 13.5

23 0.323 60 0.323 97 0.323 134 2.063 171 4.976 208 9.404 245 11.26

24 0.323 61 0.323 98 0.323 135 2.063 172 6.068 209 9.469 246 13.51

25 0.323 62 0.323 99 2.014 136 2.063 173 6.068 210 9.412 247 13.51

26 0.323 63 0.323 100 2.014 137 3.232 174 4.972 211 9.5 248 14.22

27 0.323 64 0.323 101 2.014 138 3.232 175 6.061 212 9.508 249 13.5

28 0.323 65 0.323 102 2.014 139 3.232 176 6.812 213 9.508 250 14.22

29 0.323 66 0.323 103 2.014 140 3.232 177 6.812 214 11.15 251 14.22

30 0.323 67 0.323 104 1.259 141 3.232 178 6.812 215 9.51 252 14.22

31 0.323 68 0.323 105 1.259 142 3.232 179 6.9 216 9.51 253 14.22

32 0.323 69 0.323 106 1.259 143 3.743 180 6.82 217 9.51 254 14.33

33 0.323 70 0.323 107 1.259 144 3.743 181 6.902 218 11.15 255 15.16

34 0.323 71 0.323 108 1.259 145 3.743 182 6.902 219 11.15

35 0.323 72 0.323 109 1.259 146 3.743 183 6.902 220 11.15

36 0.323 73 0.323 110 1.259 147 3.743 184 6.902 221 11.15

37 0.323 74 0.323 111 1.259 148 4.902 185 6.902 222 11.15

Appendix II

99

Appendix II

Luminance output (cd/m2) of red light after gamma correction

49 6.383 79 10.523 109 16.584 139 19.830 169 25.936 199 30.028 229 34.475

50 6.383 80 11.786 110 16.584 140 20.264 170 25.936 200 29.726 230 34.335

51 6.383 81 11.786 111 14.641 141 20.264 171 25.936 201 30.016 231 34.335

52 6.403 82 11.786 112 14.641 142 21.178 172 25.482 202 30.016 232 34.461

53 6.403 83 11.786 113 16.580 143 21.622 173 26.511 203 30.152 233 35.165

54 6.403 84 11.786 114 17.031 144 21.622 174 26.655 204 30.152 234 35.451

55 6.403 85 11.786 115 17.916 145 21.762 175 26.655 205 30.736 235 35.451

56 6.396 86 11.786 116 18.067 146 21.762 176 26.655 206 31.026 236 35.451

57 6.416 87 11.786 117 18.067 147 21.762 177 26.655 207 31.307 237 35.451

58 6.416 88 11.786 118 17.918 148 21.762 178 26.655 208 31.307 238 35.599

59 6.416 89 11.786 119 17.918 149 21.762 179 27.244 209 31.307 239 36.023

60 6.416 90 11.786 120 18.075 150 22.345 180 27.396 210 31.307 240 35.441

61 6.410 91 11.364 121 18.075 151 22.345 181 27.396 211 31.893 241 36.028

62 6.410 92 11.790 122 18.075 152 23.080 182 27.396 212 31.893 242 36.028

63 6.410 93 14.503 123 19.239 153 23.080 183 27.396 213 31.893 243 36.151

64 8.446 94 11.939 124 19.239 154 23.233 184 27.534 214 32.020 244 36.706

65 8.470 95 11.939 125 19.239 155 23.088 185 27.396 215 32.020 245 36.720

66 8.470 96 12.774 126 18.065 156 23.233 186 27.396 216 32.161 246 36.720

67 8.470 97 12.774 127 19.678 157 23.233 187 27.534 217 32.161 247 36.720

68 8.323 98 12.774 128 19.240 158 23.233 188 27.534 218 32.161 248 36.720

69 8.323 99 14.497 129 18.054 159 23.677 189 28.120 219 33.150 249 36.720

70 8.455 100 14.497 130 19.821 160 23.677 190 28.120 220 32.570 250 36.720

71 8.455 101 14.497 131 19.237 161 23.677 191 28.705 221 33.155 251 36.720

72 8.455 102 14.497 132 19.680 162 23.677 192 29.148 222 33.155 252 36.720

73 8.455 103 14.497 133 19.680 163 24.423 193 29.148 223 33.286 253 36.729

74 10.511 104 14.497 134 19.680 164 25.200 194 29.294 224 33.286 254 36.729

75 10.511 105 14.641 135 19.680 165 25.200 195 29.294 225 33.286 255 36.729

76 10.511 106 14.641 136 19.830 166 25.489 196 29.294 226 34.190

77 10.511 107 14.641 137 19.830 167 25.489 197 29.732 227 34.475

78 10.511 108 16.584 138 19.830 168 25.489 198 30.028 228 34.475

Linearization Check (Lmax + Lmin = 2*Lave)

Lmax + Lmin: 43.02518117

Lave: 45.14217367

% Error (Lmax + Lmin - Lave)/(Lmax + Lmin + Lave): -0.02401107

Appendix II

100

Luminance output (cd/m2) of green light after gamma correction

27 10.907 60 30.256 93 47.762 126 65.845 159 83.313 192 101.219 225 118.349

28 10.907 61 30.256 94 48.803 127 66.939 160 83.766 193 101.734 226 118.794

29 10.932 62 30.256 95 49.849 128 66.939 161 84.961 194 102.252 227 118.794

30 10.932 63 30.256 96 49.849 129 67.465 162 85.483 195 102.252 228 119.844

31 10.932 64 33.310 97 51.005 130 68.571 163 86.032 196 103.275 229 120.283

32 10.932 65 30.265 98 50.490 131 69.065 164 86.032 197 103.786 230 121.374

33 14.741 66 33.294 99 50.490 132 69.065 165 86.032 198 103.786 231 121.374

34 14.741 67 33.821 100 51.018 133 69.574 166 87.636 199 104.787 232 121.916

35 14.741 68 35.867 101 51.018 134 70.628 167 87.681 200 105.303 233 122.939

36 14.741 69 35.867 102 53.678 135 69.574 168 88.171 201 105.860 234 122.939

37 14.741 70 35.867 103 53.164 136 71.173 169 89.262 202 106.396 235 123.420

38 14.741 71 37.395 104 53.164 137 71.667 170 89.766 203 106.396 236 123.900

39 14.741 72 37.395 105 53.164 138 72.213 171 89.766 204 107.409 237 123.900

40 15.226 73 38.478 106 53.640 139 73.273 172 90.801 205 107.409 238 124.430

41 15.226 74 37.395 107 56.293 140 73.273 173 90.801 206 108.401 239 125.485

42 15.226 75 38.478 108 56.836 141 73.762 174 91.310 207 108.401 240 126.478

43 15.226 76 38.478 109 56.836 142 75.359 175 92.392 208 109.498 241 126.009

44 15.226 77 38.478 110 56.836 143 75.359 176 92.886 209 109.950 242 126.946

45 18.247 78 38.478 111 57.351 144 75.359 177 93.419 210 109.950 243 126.946

46 24.181 79 40.004 112 58.413 145 75.359 178 93.942 211 110.453 244 127.412

47 24.181 80 40.004 113 58.925 146 76.939 179 94.455 212 111.506 245 128.442

48 24.181 81 40.004 114 59.680 147 77.473 180 94.993 213 111.506 246 128.953

49 24.181 82 41.554 115 60.535 148 77.473 181 96.019 214 112.519 247 129.412

50 24.181 83 42.052 116 60.535 149 76.939 182 96.562 215 112.519 248 129.412

51 24.682 84 43.094 117 61.585 150 79.077 183 96.033 216 112.519 249 130.879

52 27.189 85 43.094 118 62.124 151 79.077 184 97.095 217 114.086 250 130.375

53 27.189 86 43.621 119 62.124 152 79.629 185 97.592 218 114.611 251 130.879

54 27.189 87 44.111 120 61.585 153 80.128 186 98.160 219 115.105 252 132.478

55 27.189 88 44.111 121 63.189 154 80.128 187 98.160 220 115.643 253 132.478

56 27.189 89 45.666 122 63.725 155 81.736 188 99.698 221 115.643 254 132.924

57 27.687 90 47.240 123 63.725 156 81.736 189 99.698 222 116.155 255 133.270

58 27.687 91 47.762 124 64.308 157 82.238 190 99.698 223 116.665

59 27.687 92 47.240 125 65.357 158 83.313 191 100.701 224 117.790


Lmax + Lmin: 142.032228

Lave: 147.0716378


Appendix II

101

Luminance output (cd/m2) of blue light after gamma correction

99 6.624 122 6.690 145 8.554 168 10.937 191 11.578 214 13.382 237 14.064

100 6.624 123 6.690 146 8.554 169 9.614 192 11.578 215 12.539 238 14.064

101 6.624 124 6.690 147 8.554 170 10.429 193 11.578 216 12.539 239 14.064

102 6.624 125 6.690 148 9.556 171 9.615 194 11.578 217 12.539 240 14.064

103 6.624 126 6.690 149 9.556 172 10.431 195 11.578 218 13.382 241 14.782

104 5.448 127 6.690 150 9.556 173 10.431 196 12.216 219 13.382 242 14.782

105 5.448 128 6.690 151 9.556 174 9.612 197 12.216 220 13.382 243 14.782

106 5.448 129 6.690 152 9.556 175 10.426 198 12.216 221 13.382 244 14.471

107 5.448 130 6.690 153 9.556 176 10.937 199 11.583 222 13.382 245 13.436

108 5.448 131 6.690 154 9.556 177 10.937 200 12.217 223 13.382 246 14.476

109 5.448 132 6.690 155 9.556 178 10.937 201 12.217 224 13.382 247 14.476

110 5.448 133 6.690 156 9.556 179 10.995 202 12.217 225 13.382 248 14.782

111 5.448 134 6.690 157 9.556 180 10.942 203 12.539 226 13.431 249 14.471

112 5.448 135 6.690 158 9.556 181 10.996 204 12.219 227 13.387 250 14.782

113 5.448 136 6.690 159 9.604 182 10.996 205 12.476 228 13.387 251 14.782

114 5.448 137 8.052 160 9.604 183 10.996 206 12.532 229 13.387 252 14.782

115 6.629 138 8.052 161 9.604 184 10.996 207 13.006 230 13.387 253 14.782

116 6.690 139 8.052 162 9.604 185 10.996 208 12.481 231 13.387 254 14.829

117 6.690 140 8.052 163 9.604 186 10.996 209 12.517 232 13.436 255 15.174

118 6.690 141 8.052 164 9.604 187 10.996 210 12.486 233 13.436

119 6.690 142 8.052 165 9.604 188 10.996 211 12.533 234 14.064

120 6.690 143 8.554 166 9.609 189 10.996 212 12.538 235 14.064

121 6.690 144 8.554 167 9.609 190 10.996 213 12.538 236 14.064


Lmax + Lmin: 20.71972388

Lave: 21.01680659


Appendix III

102

Appendix III

Normalized available range of red

49 0.005 79 0.150 109 0.295 139 0.440 169 0.585 199 0.729 229 0.874

50 0.010 80 0.155 110 0.300 140 0.444 170 0.589 200 0.734 230 0.879

51 0.014 81 0.159 111 0.304 141 0.449 171 0.594 201 0.739 231 0.884

52 0.019 82 0.164 112 0.309 142 0.454 172 0.599 202 0.744 232 0.889

53 0.024 83 0.169 113 0.314 143 0.459 173 0.604 203 0.749 233 0.894

54 0.029 84 0.174 114 0.319 144 0.464 174 0.609 204 0.754 234 0.899

55 0.034 85 0.179 115 0.324 145 0.469 175 0.614 205 0.758 235 0.903

56 0.039 86 0.184 116 0.329 146 0.473 176 0.618 206 0.763 236 0.908

57 0.043 87 0.188 117 0.333 147 0.478 177 0.623 207 0.768 237 0.913

58 0.048 88 0.193 118 0.338 148 0.483 178 0.628 208 0.773 238 0.918

59 0.053 89 0.198 119 0.343 149 0.488 179 0.633 209 0.778 239 0.923

60 0.058 90 0.203 120 0.348 150 0.493 180 0.638 210 0.783 240 0.928

61 0.063 91 0.208 121 0.353 151 0.498 181 0.643 211 0.787 241 0.932

62 0.068 92 0.213 122 0.357 152 0.502 182 0.647 212 0.792 242 0.937

63 0.072 93 0.217 123 0.362 153 0.507 183 0.652 213 0.797 243 0.942

64 0.077 94 0.222 124 0.367 154 0.512 184 0.657 214 0.802 244 0.947

65 0.082 95 0.227 125 0.372 155 0.517 185 0.662 215 0.807 245 0.952

66 0.087 96 0.232 126 0.377 156 0.522 186 0.667 216 0.812 246 0.957

67 0.092 97 0.237 127 0.382 157 0.527 187 0.671 217 0.816 247 0.961

68 0.097 98 0.242 128 0.386 158 0.531 188 0.676 218 0.821 248 0.966

69 0.101 99 0.246 129 0.391 159 0.536 189 0.681 219 0.826 249 0.971

70 0.106 100 0.251 130 0.396 160 0.541 190 0.686 220 0.831 250 0.976

71 0.111 101 0.256 131 0.401 161 0.546 191 0.691 221 0.836 251 0.981

72 0.116 102 0.261 132 0.406 162 0.551 192 0.696 222 0.841 252 0.986

73 0.121 103 0.266 133 0.411 163 0.556 193 0.700 223 0.845 253 0.990

74 0.126 104 0.271 134 0.415 164 0.560 194 0.705 224 0.850 254 0.995

75 0.130 105 0.275 135 0.420 165 0.565 195 0.710 225 0.855 255 1.000

76 0.135 106 0.280 136 0.425 166 0.570 196 0.715 226 0.860

77 0.140 107 0.285 137 0.430 167 0.575 197 0.720 227 0.865

78 0.145 108 0.290 138 0.435 168 0.580 198 0.725 228 0.870

Appendix III

103

Normalized available range of green

27 0.004 60 0.148 93 0.293 126 0.437 159 0.581 192 0.725 225 0.869

28 0.009 61 0.153 94 0.297 127 0.441 160 0.585 193 0.729 226 0.873

29 0.013 62 0.157 95 0.301 128 0.445 161 0.590 194 0.734 227 0.878

30 0.017 63 0.162 96 0.306 129 0.450 162 0.594 195 0.738 228 0.882

31 0.022 64 0.166 97 0.310 130 0.454 163 0.598 196 0.742 229 0.886

32 0.026 65 0.170 98 0.314 131 0.459 164 0.603 197 0.747 230 0.891

33 0.031 66 0.175 99 0.319 132 0.463 165 0.607 198 0.751 231 0.895

34 0.035 67 0.179 100 0.323 133 0.467 166 0.611 199 0.755 232 0.900

35 0.039 68 0.183 101 0.328 134 0.472 167 0.616 200 0.760 233 0.904

36 0.044 69 0.188 102 0.332 135 0.476 168 0.620 201 0.764 234 0.908

37 0.048 70 0.192 103 0.336 136 0.480 169 0.624 202 0.769 235 0.913

38 0.052 71 0.197 104 0.341 137 0.485 170 0.629 203 0.773 236 0.917

39 0.057 72 0.201 105 0.345 138 0.489 171 0.633 204 0.777 237 0.921

40 0.061 73 0.205 106 0.349 139 0.493 172 0.638 205 0.782 238 0.926

41 0.066 74 0.210 107 0.354 140 0.498 173 0.642 206 0.786 239 0.930

42 0.070 75 0.214 108 0.358 141 0.502 174 0.646 207 0.790 240 0.934

43 0.074 76 0.218 109 0.362 142 0.507 175 0.651 208 0.795 241 0.939

44 0.079 77 0.223 110 0.367 143 0.511 176 0.655 209 0.799 242 0.943

45 0.083 78 0.227 111 0.371 144 0.515 177 0.659 210 0.803 243 0.948

46 0.087 79 0.231 112 0.376 145 0.520 178 0.664 211 0.808 244 0.952

47 0.092 80 0.236 113 0.380 146 0.524 179 0.668 212 0.812 245 0.956

48 0.096 81 0.240 114 0.384 147 0.528 180 0.672 213 0.817 246 0.961

49 0.100 82 0.245 115 0.389 148 0.533 181 0.677 214 0.821 247 0.965

50 0.105 83 0.249 116 0.393 149 0.537 182 0.681 215 0.825 248 0.969

51 0.109 84 0.253 117 0.397 150 0.541 183 0.686 216 0.830 249 0.974

52 0.114 85 0.258 118 0.402 151 0.546 184 0.690 217 0.834 250 0.978

53 0.118 86 0.262 119 0.406 152 0.550 185 0.694 218 0.838 251 0.983

54 0.122 87 0.266 120 0.410 153 0.555 186 0.699 219 0.843 252 0.987

55 0.127 88 0.271 121 0.415 154 0.559 187 0.703 220 0.847 253 0.991

56 0.131 89 0.275 122 0.419 155 0.563 188 0.707 221 0.852 254 0.996

57 0.135 90 0.279 123 0.424 156 0.568 189 0.712 222 0.856 255 1.000

58 0.140 91 0.284 124 0.428 157 0.572 190 0.716 223 0.860

59 0.144 92 0.288 125 0.432 158 0.576 191 0.721 224 0.865

Appendix III

104

Normalized available range of blue

99 0.006 122 0.153 145 0.299 168 0.446 191 0.592 214 0.739 237 0.885

100 0.013 123 0.159 146 0.306 169 0.452 192 0.599 215 0.745 238 0.892

101 0.019 124 0.166 147 0.312 170 0.459 193 0.605 216 0.752 239 0.898

102 0.025 125 0.172 148 0.318 171 0.465 194 0.611 217 0.758 240 0.904

103 0.032 126 0.178 149 0.325 172 0.471 195 0.618 218 0.764 241 0.911

104 0.038 127 0.185 150 0.331 173 0.478 196 0.624 219 0.771 242 0.917

105 0.045 128 0.191 151 0.338 174 0.484 197 0.631 220 0.777 243 0.924

106 0.051 129 0.197 152 0.344 175 0.490 198 0.637 221 0.783 244 0.930

107 0.057 130 0.204 153 0.350 176 0.497 199 0.643 222 0.790 245 0.936

108 0.064 131 0.210 154 0.357 177 0.503 200 0.650 223 0.796 246 0.943

109 0.070 132 0.217 155 0.363 178 0.510 201 0.656 224 0.803 247 0.949

110 0.076 133 0.223 156 0.369 179 0.516 202 0.662 225 0.809 248 0.955

111 0.083 134 0.229 157 0.376 180 0.522 203 0.669 226 0.815 249 0.962

112 0.089 135 0.236 158 0.382 181 0.529 204 0.675 227 0.822 250 0.968

113 0.096 136 0.242 159 0.389 182 0.535 205 0.682 228 0.828 251 0.975

114 0.102 137 0.248 160 0.395 183 0.541 206 0.688 229 0.834 252 0.981

115 0.108 138 0.255 161 0.401 184 0.548 207 0.694 230 0.841 253 0.987

116 0.115 139 0.261 162 0.408 185 0.554 208 0.701 231 0.847 254 0.994

117 0.121 140 0.268 163 0.414 186 0.561 209 0.707 232 0.854 255 1.000

118 0.127 141 0.274 164 0.420 187 0.567 210 0.713 233 0.860

119 0.134 142 0.280 165 0.427 188 0.573 211 0.720 234 0.866

120 0.140 143 0.287 166 0.433 189 0.580 212 0.726 235 0.873

121 0.146 144 0.293 167 0.439 190 0.586 213 0.732 236 0.879

Appendix IV

105

Appendix IV

Emission spectra of red

400 1.19E-04 451 8.79E-05 502 7.32E-05 553 2.05E-04 604 4.41E-04 655 4.61E-04

401 1.13E-04 452 1.43E-04 503 1.23E-04 554 3.68E-04 605 4.92E-04 656 4.07E-04

402 9.64E-05 453 1.69E-04 504 1.54E-04 555 7.01E-04 606 6.48E-04 657 2.73E-04

403 7.45E-05 454 2.55E-04 505 1.16E-04 556 6.07E-04 607 7.22E-04 658 1.54E-04

404 6.94E-05 455 2.79E-04 506 1.05E-04 557 2.71E-04 608 6.67E-04 659 1.93E-04

405 9.57E-05 456 2.79E-04 507 1.15E-04 558 1.96E-04 609 6.86E-04 660 2.37E-04

406 6.28E-05 457 2.20E-04 508 1.31E-04 559 1.40E-04 610 8.67E-04 661 2.15E-04

407 8.04E-05 458 1.44E-04 509 7.42E-05 560 1.05E-04 611 1.02E-03 662 2.11E-04

408 1.44E-04 459 1.85E-04 510 1.71E-04 561 1.86E-04 612 1.10E-03 663 3.17E-04

409 6.40E-05 460 1.76E-04 511 3.18E-04 562 2.71E-04 613 1.01E-03 664 2.34E-04

410 0.00E+00 461 1.02E-04 512 3.76E-04 563 3.06E-04 614 1.46E-03 665 2.83E-04

411 0.00E+00 462 1.51E-04 513 4.76E-04 564 3.30E-04 615 6.57E-03 666 3.37E-04

412 1.13E-05 463 1.44E-04 514 4.00E-04 565 2.39E-04 616 1.66E-02 667 2.67E-04

413 1.41E-04 464 1.60E-04 515 2.42E-04 566 2.04E-04 617 1.16E-02 668 1.99E-04

414 2.18E-04 465 2.21E-04 516 1.84E-04 567 2.74E-04 618 3.14E-03 669 1.71E-04

415 1.74E-04 466 2.04E-04 517 1.71E-04 568 2.93E-04 619 2.18E-03 670 2.97E-04

416 9.02E-05 467 2.89E-04 518 1.29E-04 569 2.40E-04 620 2.99E-03 671 4.54E-04

417 3.80E-05 468 3.28E-04 519 9.38E-05 570 1.82E-04 621 3.36E-03 672 6.21E-04

418 1.42E-04 469 2.02E-04 520 1.07E-04 571 1.93E-04 622 3.33E-03 673 5.73E-04

419 8.97E-05 470 6.14E-05 521 1.21E-04 572 1.84E-04 623 4.60E-03 674 3.82E-04

420 1.95E-05 471 7.49E-05 522 1.61E-04 573 1.63E-04 624 1.24E-02 675 1.73E-04

421 1.04E-04 472 2.23E-04 523 1.29E-04 574 1.51E-04 625 3.23E-02 676 3.74E-04

422 2.37E-04 473 1.60E-04 524 1.06E-04 575 1.87E-04 626 4.18E-02 677 4.33E-04

423 1.59E-04 474 1.76E-04 525 7.85E-05 576 1.46E-04 627 2.22E-02 678 3.35E-04

424 0.00E+00 475 2.23E-04 526 9.44E-05 577 2.52E-04 628 7.03E-03 679 2.25E-04

425 1.12E-05 476 1.82E-04 527 1.58E-04 578 3.07E-04 629 5.32E-03 680 4.07E-04

426 1.61E-04 477 1.83E-04 528 1.71E-04 579 1.94E-04 630 5.62E-03 681 3.10E-04

427 2.22E-04 478 1.04E-04 529 1.25E-04 580 1.66E-04 631 4.38E-03 682 2.44E-04

428 2.77E-04 479 4.42E-05 530 1.11E-04 581 6.22E-04 632 2.73E-03 683 2.98E-04

429 2.54E-04 480 1.11E-04 531 1.44E-04 582 1.76E-03 633 1.85E-03 684 3.84E-04

430 1.35E-04 481 1.43E-04 532 9.69E-05 583 1.17E-03 634 1.42E-03 685 8.71E-04

431 9.44E-05 482 1.10E-04 533 1.47E-04 584 3.26E-04 635 9.05E-04 686 2.62E-03

432 1.57E-04 483 1.11E-04 534 1.94E-04 585 6.50E-04 636 6.68E-04 687 2.08E-03

433 1.87E-04 484 9.54E-05 535 2.32E-04 586 1.67E-03 637 6.14E-04 688 7.87E-04

434 2.43E-04 485 1.67E-04 536 2.22E-04 587 1.67E-03 638 5.58E-04 689 4.67E-04

435 1.68E-04 486 1.62E-04 537 4.59E-04 588 1.33E-03 639 4.96E-04 690 4.01E-04

436 9.83E-05 487 1.60E-04 538 1.17E-03 589 9.71E-04 640 3.27E-04 691 4.42E-04

437 8.53E-05 488 2.13E-04 539 1.39E-03 590 4.12E-04 641 2.02E-04 692 5.35E-04

438 2.36E-04 489 2.23E-04 540 7.97E-04 591 4.56E-04 642 3.21E-04 693 3.08E-04

Appendix IV

106

439 2.44E-04 490 1.51E-04 541 3.42E-04 592 4.91E-04 643 3.77E-04 694 5.18E-04

440 1.95E-04 491 7.63E-05 542 2.89E-04 593 1.52E-03 644 4.09E-04 695 8.19E-04

441 3.20E-04 492 1.16E-04 543 2.83E-04 594 5.42E-03 645 4.16E-04 696 2.08E-03

442 2.67E-04 493 1.13E-04 544 2.95E-04 595 6.33E-03 646 2.27E-04 697 1.72E-03

443 1.39E-04 494 1.81E-04 545 2.18E-04 596 3.59E-03 647 2.52E-04 698 1.23E-03

444 1.39E-04 495 3.14E-04 546 2.02E-04 597 1.09E-03 648 3.17E-04 699 9.98E-04

445 2.17E-04 496 3.54E-04 547 1.69E-04 598 6.59E-04 649 2.58E-04 700 7.97E-04

446 1.85E-04 497 2.38E-04 548 1.65E-04 599 6.18E-04 650 3.03E-04

447 1.72E-04 498 1.43E-04 549 1.42E-04 600 5.14E-04 651 3.96E-04

448 1.74E-04 499 2.06E-04 550 1.56E-04 601 5.10E-04 652 2.50E-04

449 1.48E-04 500 2.10E-04 551 2.10E-04 602 5.88E-04 653 3.29E-04

450 1.24E-04 501 9.10E-05 552 1.89E-04 603 4.53E-04 654 4.76E-04

Emission spectra of green

400 7.25E-06 451 2.13E-04 502 3.60E-03 553 4.68E-03 604 7.95E-04 655 2.77E-04

401 4.72E-05 452 2.98E-04 503 3.76E-03 554 4.59E-03 605 8.13E-04 656 3.47E-04

402 2.56E-05 453 2.90E-04 504 3.92E-03 555 4.48E-03 606 8.87E-04 657 2.95E-04

403 0.00E+00 454 3.91E-04 505 3.97E-03 556 4.40E-03 607 7.72E-04 658 2.43E-04

404 0.00E+00 455 4.04E-04 506 4.05E-03 557 4.32E-03 608 6.16E-04 659 1.84E-04

405 3.43E-05 456 3.96E-04 507 4.20E-03 558 4.28E-03 609 6.55E-04 660 7.05E-06

406 4.96E-05 457 4.23E-04 508 4.36E-03 559 4.08E-03 610 5.15E-04 661 1.06E-04

407 1.02E-05 458 2.05E-04 509 4.42E-03 560 4.01E-03 611 4.61E-04 662 1.33E-04

408 2.49E-05 459 2.72E-04 510 4.58E-03 561 3.93E-03 612 5.42E-04 663 2.49E-04

409 1.17E-04 460 3.52E-04 511 4.75E-03 562 3.88E-03 613 5.27E-04 664 1.57E-04

410 3.19E-05 461 3.25E-04 512 4.83E-03 563 3.81E-03 614 4.37E-04 665 2.42E-04

411 0.00E+00 462 3.45E-04 513 4.98E-03 564 3.66E-03 615 5.68E-04 666 1.44E-04

412 5.65E-06 463 3.23E-04 514 5.02E-03 565 3.57E-03 616 7.31E-04 667 1.07E-04

413 5.50E-05 464 4.13E-04 515 5.06E-03 566 3.38E-03 617 6.81E-04 668 1.22E-04

414 1.20E-04 465 4.95E-04 516 5.23E-03 567 3.29E-03 618 5.22E-04 669 1.51E-06

415 2.58E-04 466 4.64E-04 517 5.32E-03 568 3.18E-03 619 4.75E-04 670 2.69E-05

416 1.39E-04 467 5.52E-04 518 5.31E-03 569 3.03E-03 620 5.23E-04 671 1.24E-04

417 4.03E-06 468 6.51E-04 519 5.41E-03 570 3.03E-03 621 4.35E-04 672 2.59E-04

418 1.53E-05 469 6.99E-04 520 5.50E-03 571 3.04E-03 622 3.27E-04 673 3.12E-04

419 0.00E+00 470 7.88E-04 521 5.55E-03 572 2.89E-03 623 3.26E-04 674 2.78E-04

420 0.00E+00 471 7.89E-04 522 5.63E-03 573 2.75E-03 624 4.42E-04 675 1.56E-04

421 2.61E-05 472 8.09E-04 523 5.66E-03 574 2.66E-03 625 5.80E-04 676 1.60E-04

422 1.29E-04 473 8.45E-04 524 5.70E-03 575 2.62E-03 626 7.20E-04 677 1.54E-04

423 1.31E-04 474 8.73E-04 525 5.71E-03 576 2.53E-03 627 5.73E-04 678 1.30E-04

424 3.20E-05 475 1.07E-03 526 5.77E-03 577 2.43E-03 628 5.32E-04 679 1.21E-04

425 8.44E-05 476 1.08E-03 527 5.79E-03 578 2.38E-03 629 3.99E-04 680 4.10E-04

426 7.21E-05 477 1.10E-03 528 5.72E-03 579 2.25E-03 630 3.65E-04 681 2.81E-04

427 4.40E-06 478 1.15E-03 529 5.72E-03 580 2.16E-03 631 3.75E-04 682 4.72E-05

428 1.03E-04 479 1.24E-03 530 5.74E-03 581 2.14E-03 632 3.14E-04 683 1.74E-04

Appendix IV

107

429 1.72E-04 480 1.31E-03 531 5.71E-03 582 2.02E-03 633 2.83E-04 684 2.94E-04

430 1.44E-04 481 1.29E-03 532 5.67E-03 583 1.95E-03 634 3.78E-04 685 2.17E-04

431 1.75E-04 482 1.42E-03 533 5.71E-03 584 1.89E-03 635 2.21E-04 686 2.40E-04

432 1.84E-04 483 1.61E-03 534 5.65E-03 585 1.82E-03 636 1.52E-04 687 2.19E-04

433 1.22E-04 484 1.70E-03 535 5.65E-03 586 1.68E-03 637 2.66E-04 688 4.44E-04

434 1.57E-04 485 1.72E-03 536 5.65E-03 587 1.58E-03 638 2.06E-04 689 2.51E-04

435 1.58E-04 486 1.77E-03 537 5.56E-03 588 1.51E-03 639 2.23E-04 690 1.14E-04

436 5.69E-05 487 1.92E-03 538 5.49E-03 589 1.52E-03 640 1.66E-04 691 8.55E-05

437 1.33E-04 488 2.05E-03 539 5.47E-03 590 1.35E-03 641 8.03E-05 692 2.25E-04

438 3.27E-04 489 2.13E-03 540 5.51E-03 591 1.28E-03 642 1.98E-04 693 1.99E-04

439 3.43E-04 490 2.20E-03 541 5.42E-03 592 1.25E-03 643 2.84E-04 694 1.08E-04

440 3.33E-04 491 2.33E-03 542 5.29E-03 593 1.18E-03 644 3.34E-04 695 0.00E+00

441 4.21E-04 492 2.47E-03 543 5.21E-03 594 1.21E-03 645 2.51E-04 696 7.05E-05

442 3.80E-04 493 2.55E-03 544 5.20E-03 595 1.15E-03 646 2.87E-05 697 2.63E-04

443 2.09E-04 494 2.69E-03 545 5.20E-03 596 1.16E-03 647 9.97E-05 698 4.33E-04

444 2.42E-04 495 2.76E-03 546 5.16E-03 597 1.12E-03 648 2.88E-04 699 2.03E-04

445 2.44E-04 496 2.84E-03 547 5.08E-03 598 1.06E-03 649 1.96E-04 700 2.61E-04

446 1.20E-04 497 3.00E-03 548 5.00E-03 599 1.00E-03 650 1.35E-04

447 1.65E-04 498 3.11E-03 549 4.96E-03 600 9.43E-04 651 2.67E-04

448 2.93E-04 499 3.23E-03 550 4.84E-03 601 8.68E-04 652 1.93E-04

449 3.58E-04 500 3.38E-03 551 4.80E-03 602 9.07E-04 653 2.20E-04

450 2.97E-04 501 3.44E-03 552 4.77E-03 603 8.44E-04 654 2.57E-04

Emission spectra of blue

400 8.84E-04 451 6.32E-03 502 1.07E-03 553 1.23E-04 604 7.66E-05 655 2.40E-04

401 1.04E-03 452 6.30E-03 503 1.06E-03 554 1.57E-04 605 2.98E-05 656 2.54E-04

402 1.15E-03 453 6.12E-03 504 1.05E-03 555 1.85E-04 606 6.57E-05 657 7.06E-05

403 1.17E-03 454 6.14E-03 505 9.93E-04 556 1.32E-04 607 2.10E-05 658 0.00E+00

404 1.20E-03 455 6.18E-03 506 9.42E-04 557 1.42E-04 608 0.00E+00 659 2.49E-05

405 1.25E-03 456 6.08E-03 507 9.33E-04 558 2.25E-04 609 9.13E-05 660 3.60E-06

406 1.43E-03 457 5.85E-03 508 8.34E-04 559 1.72E-04 610 4.72E-05 661 0.00E+00

407 1.60E-03 458 5.72E-03 509 7.66E-04 560 1.16E-04 611 1.71E-06 662 1.06E-05

408 1.69E-03 459 5.60E-03 510 7.76E-04 561 1.21E-04 612 5.89E-05 663 1.27E-04

409 1.79E-03 460 5.58E-03 511 7.26E-04 562 1.82E-04 613 3.01E-05 664 5.47E-05

410 1.76E-03 461 5.50E-03 512 6.87E-04 563 1.69E-04 614 0.00E+00 665 5.57E-05

411 1.90E-03 462 5.44E-03 513 6.77E-04 564 1.88E-04 615 5.79E-05 666 5.98E-05

412 2.20E-03 463 5.44E-03 514 6.24E-04 565 1.24E-04 616 2.65E-04 667 6.13E-05

413 2.46E-03 464 5.25E-03 515 6.16E-04 566 9.28E-05 617 2.72E-04 668 7.41E-05

414 2.69E-03 465 5.14E-03 516 6.10E-04 567 1.73E-04 618 1.59E-04 669 1.04E-04

415 2.69E-03 466 4.94E-03 517 5.57E-04 568 1.22E-04 619 2.75E-05 670 0.00E+00

416 2.81E-03 467 4.78E-03 518 4.81E-04 569 7.28E-05 620 1.23E-05 671 8.15E-05

417 2.99E-03 468 4.72E-03 519 4.51E-04 570 3.55E-05 621 3.97E-05 672 2.44E-04

418 3.10E-03 469 4.62E-03 520 4.65E-04 571 6.69E-05 622 0.00E+00 673 3.13E-04

Appendix IV

108

419 3.25E-03 470 4.48E-03 521 4.60E-04 572 5.29E-05 623 8.24E-05 674 1.60E-04

420 3.41E-03 471 4.44E-03 522 4.51E-04 573 8.39E-05 624 1.33E-04 675 9.92E-05

421 3.61E-03 472 4.37E-03 523 4.18E-04 574 2.21E-05 625 2.31E-04 676 4.10E-05

422 3.90E-03 473 4.11E-03 524 3.71E-04 575 6.86E-05 626 3.22E-04 677 0.00E+00

423 4.09E-03 474 4.00E-03 525 3.25E-04 576 9.94E-05 627 2.69E-04 678 3.33E-05

424 4.17E-03 475 3.96E-03 526 3.33E-04 577 1.69E-04 628 1.89E-04 679 8.07E-05

425 4.39E-03 476 3.76E-03 527 3.17E-04 578 2.02E-04 629 1.03E-04 680 1.26E-04

426 4.64E-03 477 3.57E-03 528 2.89E-04 579 1.17E-04 630 5.25E-05 681 9.18E-05

427 4.74E-03 478 3.48E-03 529 2.66E-04 580 6.28E-05 631 1.22E-04 682 2.09E-05

428 4.88E-03 479 3.36E-03 530 2.59E-04 581 4.59E-07 632 1.44E-04 683 9.18E-05

429 5.18E-03 480 3.22E-03 531 2.96E-04 582 7.92E-05 633 4.63E-05 684 1.65E-04

430 5.30E-03 481 3.04E-03 532 2.32E-04 583 8.81E-05 634 2.04E-04 685 1.07E-04

431 5.23E-03 482 2.89E-03 533 2.72E-04 584 1.08E-04 635 8.46E-05 686 1.20E-04

432 5.35E-03 483 2.81E-03 534 2.80E-04 585 6.36E-05 636 2.40E-05 687 9.70E-05

433 5.63E-03 484 2.74E-03 535 2.92E-04 586 1.79E-05 637 1.37E-04 688 1.52E-04

434 5.87E-03 485 2.60E-03 536 2.63E-04 587 1.31E-06 638 1.29E-04 689 1.81E-04

435 5.87E-03 486 2.46E-03 537 2.30E-04 588 0.00E+00 639 1.49E-04 690 4.95E-05

436 5.85E-03 487 2.31E-03 538 2.38E-04 589 0.00E+00 640 1.37E-04 691 0.00E+00

437 5.90E-03 488 2.18E-03 539 2.52E-04 590 0.00E+00 641 4.83E-05 692 5.80E-05

438 6.16E-03 489 2.08E-03 540 2.54E-04 591 2.30E-05 642 5.17E-05 693 5.38E-06

439 6.30E-03 490 1.98E-03 541 2.03E-04 592 0.00E+00 643 3.42E-05 694 1.24E-04

440 6.24E-03 491 1.94E-03 542 2.00E-04 593 0.00E+00 644 1.12E-04 695 3.58E-08

441 6.31E-03 492 1.84E-03 543 1.82E-04 594 0.00E+00 645 3.12E-05 696 7.49E-05

442 6.42E-03 493 1.72E-03 544 2.30E-04 595 5.17E-05 646 0.00E+00 697 4.23E-04

443 6.38E-03 494 1.64E-03 545 1.84E-04 596 4.65E-05 647 8.40E-06 698 3.53E-04

444 6.35E-03 495 1.53E-03 546 1.68E-04 597 2.75E-06 648 7.96E-05 699 3.08E-04

445 6.38E-03 496 1.48E-03 547 1.47E-04 598 7.71E-05 649 6.03E-05 700 1.87E-04

446 6.38E-03 497 1.46E-03 548 1.54E-04 599 9.72E-05 650 8.18E-05

447 6.45E-03 498 1.41E-03 549 2.09E-04 600 4.56E-05 651 2.25E-04

448 6.49E-03 499 1.31E-03 550 1.51E-04 601 7.10E-05 652 9.98E-05

449 6.51E-03 500 1.25E-03 551 1.54E-04 602 1.26E-04 653 1.29E-04

450 6.41E-03 501 1.15E-03 552 1.39E-04 603 6.29E-05 654 1.79E-04

Appendix V

109

Appendix V

Smith and Pokony 10° fundamentals L cone

400 0.837656 451 28.7895 502 177.4742 553 431.2225 604 341.4695 655 51.76487

401 0.993434 452 29.85085 503 183.2358 554 433.5854 605 335.701 656 48.85433

402 1.170296 453 30.89865 504 189.1361 555 435.8561 606 329.7938 657 46.07906

403 1.370876 454 31.96616 505 195.1561 556 438.0058 607 323.7798 658 43.43504

404 1.595837 455 33.08647 506 201.2877 557 440.0005 608 317.6574 659 40.91788

405 1.846398 456 34.28706 507 207.5076 558 441.7974 609 311.3923 660 38.52804

406 2.123049 457 35.59097 508 213.7964 559 443.3686 610 305.0392 661 36.25569

407 2.426527 458 37.03627 509 220.1414 560 444.6739 611 298.5607 662 34.09509

408 2.75514 459 38.63199 510 226.5102 561 445.7621 612 292.0273 663 32.04067

409 3.108878 460 40.40214 511 232.9723 562 446.7042 613 285.42 664 30.09456

410 3.486306 461 42.16824 512 239.5872 563 447.4804 614 278.757 665 28.24771

411 3.880381 462 43.80783 513 246.3298 564 448.0881 615 272.0599 666 26.49607

412 4.282559 463 45.40005 514 253.1591 565 448.5151 616 265.3326 667 24.8424

413 4.690377 464 47.00136 515 260.0439 566 448.7486 617 258.5892 668 23.27806

414 5.103503 465 48.68145 516 266.9443 567 448.7846 618 251.8465 669 21.80094

415 5.521699 466 50.488 517 273.8113 568 448.6004 619 245.1112 670 20.40711

416 5.945178 467 52.4744 518 280.5898 569 448.1945 620 238.3949 671 19.09027

417 6.375878 468 54.68046 519 287.2306 570 447.553 621 231.7476 672 17.84494

418 6.820721 469 57.11814 520 293.67 571 446.6229 622 225.2137 673 16.66982

419 7.28352 470 59.8425 521 299.873 572 445.3859 623 218.7512 674 15.56313

420 7.771571 471 62.65347 522 305.8614 573 443.8836 624 212.3387 675 14.52264

421 8.270079 472 65.28767 523 311.6525 574 442.1589 625 205.9524 676 13.54624

422 8.761558 473 67.77269 524 317.2774 575 440.26 626 199.5622 677 12.63125

423 9.245043 474 70.14917 525 322.7556 576 438.2302 627 193.1635 678 11.77504

424 9.723182 475 72.47854 526 328.1169 577 436.1185 628 186.7384 679 10.97529

425 10.20408 476 74.80813 527 333.3905 578 433.974 629 180.2773 680 10.22874

426 10.69172 477 77.18586 528 338.6097 579 431.8437 630 173.7696 681 9.531138

427 11.18384 478 79.6815 529 343.8061 580 429.7843 631 167.2196 682 8.877765

428 11.6975 479 82.34861 530 349.0158 581 427.839 632 160.6595 683 8.266644

429 12.2423 480 85.25971 531 354.2311 582 425.9359 633 154.1392 684 7.695556

430 12.81781 481 88.41975 532 359.4026 583 424.0551 634 147.698 685 7.161814

431 13.42207 482 91.73803 533 364.5091 584 422.1374 635 141.3731 686 6.663744

432 14.03691 483 95.13707 534 369.5296 585 420.0925 636 135.1975 687 6.199348

433 14.6534 484 98.55897 535 374.4407 586 417.9616 637 129.1954 688 5.766375

434 15.28541 485 101.9659 536 379.2146 587 415.6014 638 123.3923 689 5.362894

435 15.93091 486 105.3323 537 383.8297 588 412.9954 639 117.8016 690 4.987493

436 16.59889 487 108.6388 538 388.259 589 410.0927 640 112.4378 691 4.637557

437 17.28759 488 111.8801 539 392.4724 590 406.8934 641 107.2876 692 4.311376

438 18.00295 489 115.0601 540 396.4464 591 403.4043 642 102.3161 693 4.007498

439 18.74815 490 118.1841 541 400.1166 592 399.7021 643 97.52402 694 3.724434

Appendix V

110

440 19.52597 491 121.6832 542 403.4646 593 395.7784 644 92.89537 695 3.460658

441 20.29756 492 125.8264 543 406.5354 594 391.6778 645 88.43013 696 3.21548

442 21.03224 493 130.4245 544 409.3665 595 387.4037 646 84.12286 697 2.987084

443 21.74611 494 135.3268 545 412.0009 596 382.9259 647 79.96697 698 2.774744

444 22.45491 495 140.4117 546 414.492 597 378.285 648 75.95702 699 2.577441

445 23.17925 496 145.5838 547 416.8803 598 373.4504 649 72.08971 700 2.393908

446 23.93686 497 150.7755 548 419.2175 599 368.4582 650 68.3598

447 24.74793 498 155.9576 549 421.5463 600 363.3093 651 64.76617

448 25.62951 499 161.1407 550 423.9272 601 358.0236 652 61.30915

449 26.59574 500 166.4126 551 426.3598 602 352.6299 653 57.99059

450 27.68262 501 171.863 552 428.8049 603 347.102 654 54.80858

Smith and Pokony 10° fundamentals M cone

400 0.531353 451 34.69349 502 155.9674 553 249.5734 604 73.03554 655 3.686009

401 0.636209 452 35.90517 503 159.8635 554 248.1121 605 69.9189 656 3.452017

402 0.757136 453 37.08395 504 163.7984 555 246.5381 606 66.88077 657 3.232322

403 0.896008 454 38.26625 505 167.7639 556 244.8391 607 63.92835 658 3.025489

404 1.054211 455 39.48659 506 171.7612 557 243.0015 608 61.06038 659 2.829768

405 1.233258 456 40.77554 507 175.7794 558 241.0066 609 58.26749 660 2.643999

406 1.434708 457 42.15856 508 179.8108 559 238.8479 610 55.56056 661 2.468476

407 1.66124 458 43.67203 509 183.8543 560 236.5113 611 52.93539 662 2.303457

408 1.911812 459 45.33081 510 187.8952 561 234.0025 612 50.40463 663 2.149727

409 2.189168 460 47.1594 511 191.9574 562 231.3392 613 47.96969 664 2.005164

410 2.49406 461 48.98316 512 196.0519 563 228.5208 614 45.62454 665 1.869951

411 2.823967 462 50.66705 513 200.1555 564 225.5567 615 43.37361 666 1.742651

412 3.174457 463 52.29792 514 204.2337 565 222.4525 616 41.21436 667 1.624407

413 3.545261 464 53.93746 515 208.2633 566 219.2137 617 39.14298 668 1.513412

414 3.937394 465 55.65462 516 212.2157 567 215.8514 618 37.16785 669 1.409651

415 4.351778 466 57.49598 517 216.0582 568 212.368 619 35.27891 670 1.312623

416 4.789564 467 59.49763 518 219.757 569 208.7783 620 33.47882 671 1.221866

417 5.252231 468 61.67713 519 223.286 570 205.091 621 31.77849 672 1.136912

418 5.745833 469 64.06349 520 226.6116 571 201.2735 622 30.19561 673 1.057561

419 6.272704 470 66.64264 521 229.6858 572 197.3061 623 28.70796 674 0.983483

420 6.838525 471 69.18299 522 232.4945 573 193.2193 624 27.30142 675 0.914369

421 7.433503 472 71.52651 523 235.0633 574 189.0429 625 25.96369 676 0.850007

422 8.038925 473 73.71528 524 237.4159 575 184.8063 626 24.67349 677 0.790083

423 8.658489 474 75.77011 525 239.5804 576 180.5382 627 23.42875 678 0.734347

424 9.292277 475 77.74092 526 241.5887 577 176.2666 628 22.2165 679 0.682598

425 9.942427 476 79.66643 527 243.473 578 172.0189 629 21.02861 680 0.634473

426 10.61453 477 81.58957 528 245.2682 579 167.8225 630 19.85667 681 0.58979

427 11.30537 478 83.57305 529 247.018 580 163.6994 631 18.70852 682 0.548209

428 12.02863 479 85.66783 530 248.7551 581 159.6547 632 17.59724 683 0.50959

429 12.79246 480 87.94209 531 250.4784 582 155.6533 633 16.52733 684 0.473626

Appendix V

111

430 13.59798 481 90.44209 532 252.1369 583 151.6903 634 15.50256 685 0.440203

431 14.43895 482 93.11168 533 253.7102 584 147.7547 635 14.52482 686 0.409083

432 15.29529 483 95.86103 534 255.1782 585 143.8097 636 13.59657 687 0.380146

433 16.15955 484 98.61961 535 256.5234 586 139.8899 637 12.71875 688 0.353255

434 17.04313 485 101.3399 536 257.7189 587 135.9528 638 11.89126 689 0.328151

435 17.94735 486 103.9867 537 258.7454 588 132.004 639 11.11366 690 0.304953

436 18.87214 487 106.5307 538 259.582 589 128.0398 640 10.3851 691 0.283199

437 19.82469 488 108.9583 539 260.2043 590 124.0757 641 9.700427 692 0.26303

438 20.80659 489 111.2635 540 260.5934 591 120.1099 642 9.053539 693 0.244257

439 21.82148 490 113.4444 541 260.7151 592 116.1754 643 8.444426 694 0.226801

440 22.87275 491 115.9059 542 260.5705 593 112.2693 644 7.871333 695 0.210546

441 23.91794 492 118.911 543 260.1906 594 108.4095 645 7.33426 696 0.1955

442 24.92354 493 122.2869 544 259.6067 595 104.5988 646 6.832517 697 0.181499

443 25.90284 494 125.8969 545 258.8436 596 100.8314 647 6.36448 698 0.168517

444 26.87013 495 129.6329 546 257.9371 597 97.12194 648 5.929458 699 0.156479

445 27.84374 496 133.4102 547 256.9148 598 93.46412 649 5.52664 700 0.145316

446 28.84007 497 137.17 548 255.8071 599 89.8689 650 5.153757

447 29.87789 498 140.888 549 254.6394 600 86.33888 651 4.811241

448 30.9736 499 144.5717 550 253.4475 601 82.88495 652 4.49604

449 32.14244 500 148.2991 551 252.2232 602 79.51779 653 4.205618

450 33.416 501 152.1122 552 250.9392 603 76.23288 654 3.936418

Smith and Pokony 10° fundamentals S cone

400 58.89888 451 1356.831 502 132.5714 553 1.412709 604 0 655 0

401 70.3547 452 1345.806 503 124.4499 554 1.049774 605 0 656 0

402 83.54363 453 1332.863 504 116.6057 555 0.747099 606 0 657 0

403 98.62258 454 1318.072 505 109.0511 556 0.486881 607 0 658 0

404 115.7216 455 1301.569 506 101.8089 557 0.278707 608 0 659 0

405 134.9846 456 1283.353 507 94.91721 558 0.126 609 0 660 0

406 156.5211 457 1263.495 508 88.40286 559 0.032185 610 0 661 0

407 180.42 458 1242.129 509 82.32129 560 0 611 0 662 0

408 206.7363 459 1219.326 510 76.72593 561 0 612 0 663 0

409 235.4766 460 1195.201 511 71.70372 562 0 613 0 664 0

410 266.6316 461 1170.364 512 67.24304 563 0 614 0 665 0

411 299.9148 462 1145.164 513 63.24732 564 0 615 0 666 0

412 335.01 463 1119.416 514 59.63646 565 0 616 0 667 0

413 371.7692 464 1092.641 515 56.32211 566 0 617 0 668 0

414 410.0554 465 1064.77 516 53.23784 567 0 618 0 669 0

415 449.7387 466 1035.53 517 50.30149 568 0 619 0 670 0

416 490.7024 467 1004.783 518 47.43361 569 0 620 0 671 0

417 532.844 468 972.3246 519 44.55204 570 0 621 0 672 0

418 576.1155 469 938.1539 520 41.57254 571 0 622 0 673 0

419 620.4895 470 902.2439 521 38.66084 572 0 623 0 674 0

Appendix V

112

420 665.9811 471 864.4712 522 36.0258 573 0 624 0 675 0

421 711.422 472 825.1646 523 33.63795 574 0 625 0 676 0

422 755.3851 473 785.1732 524 31.46856 575 0 626 0 677 0

423 797.8417 474 745.0449 525 29.47995 576 0 627 0 678 0

424 838.7918 475 705.4644 526 27.64336 577 0 628 0 679 0

425 878.2353 476 666.8631 527 25.91154 578 0 629 0 680 0

426 916.3778 477 629.6108 528 24.23039 579 0 630 0 681 0

427 953.63 478 594.0226 529 22.56294 580 0 631 0 682 0

428 990.266 479 560.345 530 20.85235 581 0 632 0 683 0

429 1026.765 480 528.7388 531 19.19381 582 0 633 0 684 0

430 1063.798 481 498.7213 532 17.70988 583 0 634 0 685 0

431 1100.585 482 469.7891 533 16.38003 584 0 635 0 686 0

432 1135.988 483 442.0485 534 15.1844 585 0 636 0 687 0

433 1169.748 484 415.5611 535 14.09559 586 0 637 0 688 0

434 1201.659 485 390.3679 536 13.09786 587 0 638 0 689 0

435 1231.584 486 366.51 537 12.14807 588 0 639 0 690 0

436 1259.455 487 343.9943 538 11.23251 589 0 640 0 691 0

437 1285.065 488 322.8071 539 10.31558 590 0 641 0 692 0

438 1308.28 489 302.9415 540 9.365104 591 0 642 0 693 0

439 1329.029 490 284.3592 541 8.42832 592 0 643 0 694 0

440 1347.162 491 267.0822 542 7.570971 593 0 644 0 695 0

441 1362.104 492 250.9041 543 6.789632 594 0 645 0 696 0

442 1373.471 493 235.5759 544 6.075403 595 0 646 0 697 0

443 1381.483 494 220.9722 545 5.422119 596 0 647 0 698 0

444 1386.277 495 207.0485 546 4.814031 597 0 648 0 699 0

445 1388.262 496 193.8185 547 4.26141 598 0 649 0 700 0

446 1387.646 497 181.3417 548 3.734127 599 0 650 0

447 1384.838 498 169.7223 549 3.228071 600 0 651 0

448 1380.045 499 159.0876 550 2.730918 601 0 652 0

449 1373.676 500 149.6266 551 2.252254 602 0 653 0

450 1366.007 501 140.9634 552 1.811938 603 0 654 0

cone signals and activity in myopia and emmetropia · abstract iii participant group, he testimated...

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