LENS AUTOFLUORESCENCE

In aging and cataractous human lenses. Clinical applicability

SEPP O
SIIK
Department of Ophthalmology

OULU 1999

SEPPO SIIK

LENS AUTOFLUORESCENCE
In aging and cataractous human lenses. Clinical
applicability

Academic dissertation to be presented with the assent of

the Faculty of Medicine, University of Oulu, for public
discussion in Autorium 5 of the University Hospital of
Oulu, on June 11th, 1999, at 12 noon.

O U L U N Y L I O P I S TO , O U L U 1 9 9 9

Copyright 1999
Oulu University Library, 1999

Manuscript received 17.5.1999

Accepted 21.5.1999

Communicated by
Docent Eero Aarnisalo
Professor Michael Larsen

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(URL: http://herkules.oulu.fi/isbn9514252675/)
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OULU UNIVERSITY LIBRARY

OULU 1999

Siik, Seppo, Lens autofluorescence: in aging and cataractous human lenses, clinical
applicability
Department of Ophthalmology, University of Oulu, P.O. Box 5000, FIN-90401 Oulu, Finland
Acta Univ. Oul. D 525, 1999
Oulu, Finland
(Manuscript received 17.5.1999)

Abstract
This study was carried out to investigate in vivo the changes of the human lens autofluorescence
(AF) with aging and cataractogenesis. Measurements were performed in the blue-green AF range
(495 nm/ 520 nm) using a fluorometer designed, built and now clinically tested in our department.
43 random eyes of 43 healthy volunteers aged 6-86 years, five of each decade, were studied for
effects of aging and 84 eyes of 84 patients with cortical, nuclear, posterior subcapsular or mixed
lens opacities were studied for differences of various cataract types. The results were compared
with the back light scatter values obtained by the commercially available Interzeag Lens Opacity
Meter 701. Also AF and back light scatter of the lens were measured from 122 smoking males aged
57 to 76 years who participated in a cancer prevention study. The results were compared with the
widely used subjective lens opacities classification system, LOCS III. In addition data was collected
from 30 randomly chosen eyes of as many subjects with varying degrees of yellow-brown lens coloration in an otherwise healthy eye. We studied the influence of lens yellowing expressed by means
of lens AF on visibility of retinal nerve fiber layer in black-and-white images.
Lens AF profile consists of anterior and posterior peaks and a central plateau. The height of the
anterior peak was used as a measure of the maximum AF value. The square root of the ratio
between the posterior and the anterior AF peaks was used for estimating the lens transmission. Our
technique was highly reproducible. The coefficient of variation was 3.9% for maximum AF and
2.9% for the lens transmission index.
Both the maximum AF and light scatter were exponentially increased with age (r = 0.95 and
0.94, respectively; p<0.0001). According to the regression line of AF begins to increase in early
childhood. It appears by extrapolation to be absent at birth. In contrast light scatter in the lens was
present even in young children. The lens transmission for blue-green light, determined from the lens
AF curve, was almost unchanging with age up to 60 years. Thereafter it decreased rapidly and the
interindividual variation increased.
In cataractous lenses the mean AF and scatter values differed statistically significantly from
those of age matched healthy controls. The highest AF values were measured in nuclear cataracts
where AF was also related to visual acuity and an increasing yellow-brown colour of the nucleus.
About half of the total variation of the transmission index values could be accounted for by changes
in nuclear colour as assessed by the LOCS III grading system. The transmission index provided a
more precise prediction about nuclear colour and opalescence than age or light scatter did.
In cortical cataracts the AF curve was low and flattened and the maximum AF value was significantly lower than in the age matched control eyes. The highest light scatter values were measured
from cortical cataracts, but the correlation between LOCS III cortical grades and light scatter values
was rather weak.
Posterior subcapsular cataracts cannot be quantified either with AF or with light scatter measurements. Lens yellowing, expressed as lens AF, had an actual effect on retinal nerve fiber layer visibility.
AF measurements provided a better prediction about the visibility score than age or visual acuity did.
The results of the present study indicate that the lens autofluorescence measurement may be a
useful additional tool together with a subjective grading system in the follow-up of optical changes
occurring in the nuclear region of the lens.

Keywords: light transmission, light scatter, nuclear cataract, retinal nerve fiber layer
photography

Acknowledgements
This study was carried out at the Department of Ophthalmology, University of Oulu,
during the ten years 19891999.
Since Jyrki Helve and Heikki Nieminen published the article Autofluorescence of the
human diabetic lens in vivo in 1976, had Heikki Nieminen designed and constructed the
device for more accurate measurement of autofluorescence from the human lens, side by
side with his main work as a skilful photographer in our clinic. It was enjoyable and very
interesting to collaborate with him. My sincerest respect goes to Heikki, who passed away
during the final phase of this work.
I wish to express my deepest gratitude to my supervisor, Professor P. Juhani Airaksinen, M.D., Head of the Department of Ophthalmology, for providing me with the opportunity to work with this subject and for his patient guidance and support during all stages
of this study. I am deeply indebted to him for teaching me how to write scientific papers
and for his expert revision of the English of them. I am very grateful for his encouragement to the presentations of my work in ARVO and his fatherly care for me during those
two weeks in warm Sarasota in Florida May 1990 and 1992. These weeks gave me many
unforgettable moments also during leisure time and probably changed the rest of my life.
I am grateful to Professor Michael Larsen, M.D., and Docent Eero Aarnisalo, M.D.,
the official reviewers of the manuscript, for their constructive comments and criticism on
this thesis.
I acknowledge Professor Anja Tuulonen, M.D., for her scientific advice and guidance
during the study.
I wish to thank Professor Leo T. Chylack Jr., M.D., Judith Friend, M.D., John Wolfe,
M.D. and Jaakko Teikari, M.D. for their co-operation. It has been a pleasure to work with
them.
With pleasure I thank all the staff in the Eye Clinic of the Oulu University Hospital for
their positive attitude and help, and especially those who participated in this study as
healthy controls. I wish to address my special thanks to Hannu Alanko, M.D., for his
valuable help and advice with statistics and computers, Rauno Miettinen, M.D., for his
empathy and friendly teaching under my specialization in the field of Ophthalmology and
for his many kind words in after years and to Mrs Toini Pylvs-Koski for her practical
help during this work. The excellent technical assistance of Mrs Riitta Kauppi and Mrs

Liisa Krki with the staff of the Laboratory of Photography is deeply acknowledged. The
language of the manuscript of this thesis was revised by Ms Erja Vlipirtti for which I am
thankful.
I am sincerely grateful to Professor Erkki Visnen, M.D.,Ph.D., for his valuable time
and understanding support, which carried me forward throughout that period, when everything in my life looked just black.
This research was financially supported by grants from the Eye Foundation, the Eye
and Tissue Bank Foundation, the Friends of Blind and the Eye Clinic of the Oulu University Hospital, which are gratefully acknowledged.
This study could not have been completed without the support, understanding and
encouragement of my dear wife Rauni. My loving thoughts go also to our children
Tommi, Tero and Tuomo as well as to our sunny one-year-old daughter Taru. You all
make my life worth living thank you. I dedicate this work to you.
Oulunsalo, May 1999

Seppo Siik

Abbreviations
A50
A/D
AF
D/A
Fa
Fp
F/V
LOCS
LOM
NAD
NADP
NS
RNFL
SD
T
UV
V/F
W50
W75
3HK
3HKG

area of lens autofluorescence curve above half height of anterior peak

analog/digital
autofluorescence
digital/analog
value of anterior lens autofluorescence peak
value of posterior lens autofluorescence peak
frequency/voltage
Lens Opacification Classification System
Lens Opacity Meter
nicotinamide-adenine dinucleotide
nicotinamide-adenine dinucleotide phosphate
not significant
retinal nerve fiber layer
standard deviation
transmission index
ultraviolet
voltage/frequency
width of lens autofluorescence curve at height of 50% of anterior peak
width of lens autofluorescence curve at height of 75% of anterior peak
3-hydroxykynurenine
3-hydroxykynurenine glucoside

List of original articles

The thesis is based on the following articles, which are referred to in the text by their
Roman numerals:
I

Siik S, Airaksinen PJ, Tuulonen A, Alanko HI & Nieminen H (1991) Lens autofluorescence in healthy individuals. Acta Ophthalmol 69: 187-192.

II

Siik S, Airaksinen PJ & Tuulonen A (1992) Light scatter in aging and cataractous
human lens. Acta Ophthalmol 70: 383-388.

III

Siik S, Airaksinen PJ, Tuulonen A & Nieminen H (1993) Autofluorescence in cataractous human lens and its relationship to light scatter. Acta Ophthalmol 71: 388392.

IV

Siik S, Airaksinen PJ, Tuulonen A & Nieminen H (1997) Influence of lens autofluorescence on retinal nerve fiber layer evaluation. Acta Ophthalmol Scand 75: 524527.

Siik S, Chylack LT Jr, Friend J, Wolfe J, Teikari J, Nieminen H & Airaksinen PJ

Lens autofluorescence and light scatter in relation to the lens opacities classification
system, LOCS III. Submitted for publication.

Contents
Abstract
Acknowledgements
Abbreviations
List of original articles
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. What is autofluorescence? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Why measure lens autofluorescence? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Review of the literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Aging human crystalline lens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1. Light transmission in the lens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2. Classification of lens opacities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3. Quantification of lens opacities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. History of lens autofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Lens pigments and fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1. Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2. Modification by oxidative damage . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.3. Glycosylation of lens proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Other recent studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Instrumentation development of ocular fluorometry . . . . . . . . . . . . . . . . . . . . .
2.6. Analysis of lens fluorometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Purpose of the present study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1. Lens autofluorescence and light scatter in healthy individuals
(I and II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2. Lens autofluorescence and light scatter in cataractous lens
(II and III) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3. Lens autofluorescence and retinal nerve fiber layer evaluation (IV) . . .
4.1.4. Lens autofluorescence, light scatter and LOCS III (V) . . . . . . . . . . . . .
4.2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1. Description of the instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1.1. Fluorometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1.2. Lens Opacity Meter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15
15
16
17
17
18
20
20
21
22
23
23
26
28
29
29
31
32
32
32
32
33
33
34
34
34
36

4.2.2. Our analysis of lens fluorometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.3. Evaluation of lens opacities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.4. Evaluation of retinal nerve fiber layer . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.5. Statistical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.6. Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Lens autofluorescence and light scatter in healthy individuals . . . . . . . . . . . . .
5.2. Lens autofluorescence and light scatter in cataractous lenses . . . . . . . . . . . . . .
5.3. Lens autofluorescence and retinal nerve fiber layer evaluation . . . . . . . . . . . . .
5.4. Lens autofluorescence, light scatter and LOCS III . . . . . . . . . . . . . . . . . . . . . .
6. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Lens autofluorescence and light scatter in healthy individuals . . . . . . . . . . . . .
6.2. Lens autofluorescence in cataractous lenses . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Light scatter in cataractous lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4. Clinical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.1. In evaluation of retinal nerve fiber layer . . . . . . . . . . . . . . . . . . . . . . . .
6.4.2. In correction of perimetry results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.3. In epidemiological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.4. In the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5. Improvement topics: Reduction of sources of error and limitations of
the method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Original articles

36
37
38
39
39
40
40
42
46
50
56
56
58
60
61
62
64
65
65
66
69
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1. Introduction
1.1. What is autofluorescence?
The quantum theory explains that excitation radiation must be at specific frequencies to
excite the natural resonant energy levels of the substance involved. When such resonance
occurs, energy is absorbed from the incident radiation elevating free electrons into a
higher energy state. This energy can then be re-emitted by spontaneous decay of the electrons into their lower energy state; if this decay occurs in the visible spectrum, it is termed
luminescence. Luminescence refers to the emission of light due to any cause other than
high temperature.
Energy and wavelength are reciprocally related; E = h / , where E is energy of electromagnetic radiation, h is Plancks constant and is wavelength. The second law of
thermodynamics dictates that the emitted energy must be less than the absorbed energy
because vibrational energy is lost in the process. That is why the luminescence always
entails a shift from a shorter wavelength (corresponding to higher energy) in the excitation radiation to a longer wavelength (corresponding to lower energy) in the emitted light.
(Schatz et al. 1978)
Fluorescence is luminescence that is maintained only by continuous excitation; that is,
emission occurs immediately after excitation and stops when excitation stops. Fluorescence does not have an afterglow. Phosphorescence is luminescence that persists (afterglow) for some time after the excitation is removed. Modern quantum theory states that
fluorescence is actually phosphorescence in which the decay curve is so rapid that it
appears to be instantaneous. The spectral shift towards the red makes it possible to excite
a fluorophore by intense illumination over a narrow spectral band and detect the weaker
fluorescence in the exclusion of reflected and scattered excitation by the use of a suitable
narrow band filter.
Fluorescence in the eye can be either intrinsic (tryptophan-related), resulting from
amino acid residues in proteins or extrinsic (non-tryptophan), resulting from other chromophores (Pirie 1968, Lerman & Borkman 1976, Yappert et al. 1992 & 1993). They both
are visible in the lens, but can also be found in the cornea and in the retinal pigment epithelium. The extrinsic fluorophores indicate changes in the pathologic state of the lens as
a function of age and are useful indicators of redox potential, protein degradation,

16
lipofuscin accumulation and other physiological parameters. The term endogenous fluorophores is used of the fluorophores which are usually present in the eye (Cunha-Vaz
1993). In contrast to that is the term exogenous fluorophores, whose presence is motivated by for instance i.v. injection into the body for diagnostic or therapeutic purposes.
The term extrinsic is sometimes used confusingly as a synonym to the artificially introduced fluorescence.
The term autofluorescence (AF) is often used instead of fluorescence to distinguish
this fluorescence from the exogenous, artificial fluorescence in the eye. I have used both
terms in my thesis.

1.2. Why measure lens autofluorescence?

Age-related and cataractous changes in the human lens interfere with retinal imaging by
causing scatter and absorption of light as well as AF, particularly at short wavelengths
(Lerman & Borkman 1976). The results of many psychophysical, optical and electrophysiological tests used in ophthalmic diagnosis and in vision research depend on the
amount of light reaching the retina. Among others this problem was recognized when
blue-on-yellow perimetry was introduced (Johnson et al. 1988). Therefore, if one could
correct for the loss of light in the lens of a given individual, the data would be improved.
On the other hand cataract is a major cause of visual disability worldwide (Thylefors
1997). There are more efficient surgical techniques, but a less costly and more efficient
approach might be identifying and eliminating environmental and nutritional risk factors
for cataract. Attempts to influence the cataract by medical treatment have also a long history (Kador 1983, Bron et al. 1987). Better understanding of the molecular biology of cataractogenesis might enable us to find medical or dietary means that retard cataract formation. It should be noted that if cataract formation could be delayed by 10 years, the number of individuals needing cataract surgery would be reduced by 45% over that 10 year
period (Kupfer 1984) and the number of cataract blind in the world would decrease by
approximately 10 million.
There is demand for objective, accurate and reproducible methods to quantify the
transmission properties of the lens and to monitor development of lens opacities in longitudinal studies, when the effect of various risk factors or anticataract medications on the
course of age-related cataractogenesis is assessed. It is important to distinguish true
change in cataract status beyond variation due to measurement error, grader error or other
noise in the assessment. Among the three main types of cataract, the grading of nuclear
cataract is considered the most problematic (Sasaki et al. 1997). On the other hand, it has
been proposed that the decreased transmission of visible light in the aging lens and in
nuclear cataract is mainly due to the accumulation of lens fluorogens (Lerman & Borkman 1976).
The lens autofluorescence measurement is a rapid, objective and noninvasive method,
which may provide information on the transmission properties in the central region of the
lens.

2. Review of the literature

2.1. Aging human crystalline lens
The adult human lens is a pale yellow ovoid, approximately 9-10 mm in diameter and 4
5 mm in thickness, weighing about 250 mg. The lens is composed of epithelial and fiber
cells which are completely surrounded by a collagenous capsule. The wet weight of the
lens increases as cellular proliferation and differentiation continue into advanced age.
Protein constitutes 2050% of the lens wet mass. This is the highest concentration of protein of any tissue in the body. About 8090% of the lens protein is water-soluble and can
be fractionated into -, - and -crystallins. The water-insoluble so-called albuminoid
fraction is composed of membrane-bound proteins and aggregated crystallins. (Spector et
al. 1973, Berger et al. 1980, Perkins 1988, Chylack 1994) Lipids in the lens are concentrated in the plasma membranes of the cortical and nuclear lens fibers. The most prominent lipids are sphingomyelin and cholesterol. In the human lens glycolipids are also
present. (Broekhuyse 1973)
In the adult lens active metabolism occurs in the epithelium and superficial cortex. The
nucleus is almost totally devoid of active metabolism. About 80% of the glucose used by
the lens is metabolized by anaerobic glycolysis to produce lactic acid and adenosine triphosphate (ATP). Glucose can also be oxidized through the hexose monophosphate shunt to
produce NADPH for reductive needs. (Berger et al. 1980) Profound changes in cellular
and molecular composition occur in the lens with aging, some of them as precursors of
cataract formation. However, significant changes in lipid composition have not been
found during cataractogenesis (Broekhuyse 1973). It has been estimated that more than
75% of persons older than 75 years have lens opacities (Sperduto & Hiller 1984). Cataract is commonly defined as any opacity in the ocular lens being the most common cause
of treatable blindness in the world.
A wide variety of insults of environmental, nutritional, metabolic and genetic nature
are believed to adversely affect the homeostasis of the normal lens and thus the maintenance of lens transparency. However, two fundamental processes seem to be involved.
One process that usually occurs in the lens cortex is caused by an imbalance in electrolytes, increase of internal sodium (Na+) and free calcium (Ca2+) and decrease of potassium (K+), leading to overhydration of the lens and liquefaction of the lens fibers

18
(Duncan et al. 1989 & 1997). Increase in calcium content in the lens is known to cause
protein aggregation and light scattering as well as activation of proteolytic enzymes
(calpain) following degradation of proteins (Hightower et al. 1987). The second process
that usually occurs in the lens nucleus is characterized by the physical and chemical
changes of the lens structural proteins, the crystallins, leading to their aggregation, many
of them insoluble (Dilley 1975, Spector 1984). The coloration that lens acquires with
aging indicates that these changes in proteins involve amino acid residues that lead to colored products. Because the crystallins do not rapidly turn over in the lens, they are subjected to various changes due to oxidation, nonenzymatic glycosylation, proteolysis, deamidation and phosphorylation. Two main mechanisms being proposed to account for
these age-related changes, non-enzymatic glycosylation of lens proteins and formation of
oxidative, free radicals and their reaction with tissue components are discussed more
widely in chapter 2.3.

2.1.1. Light transmission in the lens

When electromagnetic radiation passes through a medium (lens) some of the energy
passes directly through. Some is scattered by the medium, resulting in a general illumination within the eye. Some is absorbed when a part of the energy is dissipated into heat as
the result of energy-absorbing electronic transitions and the rest is reradiated at longer
wavelengths (autofluorescence). A small amount is reflected by surfaces separating media
of different refractive indices. (Boettner and Wolter 1962)
The physics of scattering for large and small particles (compared to the wavelength of
light) is different. The scattering by large particles consists of a mixture of diffraction,
diffuse reflection and refraction, while that for small particles is dependent upon the volume of the particle and the wavelength of light (Longhurst 1967). Light scatter may occur
at any angle. Scatter occurring at angles of greater than 90 to the incident source is
termed backscatter, while the scatter at angles of less than 90 is termed forward scatter.
Reduction of the retinal contrast is caused by an increase in forward scatter, not backscatter. Forward scatter cannot be directly derived from backscatter (Bettelheim & Chylack
1985), i.e. for example from the slit-lamp image. However, within nuclear cataract, which
is optically most regular, a good correlation between forward and backscatter has been
found (de Waard et al. 1992). A correlation between contrast sensitivity and backscatter
has also been publicized (Rouhiainen et al. 1996). The measurement of back scattering, as
we have done in this study, also includes a certain amount of reflected light, especially
within structures such as the lens, where the refractive index is not constant.
The transmission in the lens of ultraviolet and short wavelength visible radiation
decreases with age (Said & Weale 1959, Boettner & Wolter 1962). In addition to increasing absorption the light scattering in the older lens is much higher (Spector et al. 1973,
Weale 1987). The degree of color of the lens plays a significant role in the transmission of
visible light in normal aged human lenses as well as in moderate cortical and advanced
brown nuclear cataracts. However, the transmission of light can be markedly impaired in
those lenses in which advanced cortical opacities develop, although the lens may not
show any significant change in the degree of yellowness. (Lerman & Borkman 1976)

19
Except for the lens, the preretinal media show only a little change in transmission of light
with age (van den Berg & Tan 1994).
There is a high degree of short-range spatial order, similar to that seen in dense liquids
or glasses, between the proteins in the lens (Trukel 1962, Delaye & Tardieu 1983). In
1971, Benedek suggested that the condensation of lens proteins into randomly distributed
high molecular weight aggregates could produce enough fluctuation in protein density i.e.
in the index of refraction to explain increasing scattering. The increase in size and concentration of the large -crystallin macromolecules with aging has been assumed to be
involved (Spector et al. 1973). They measured back scatter of light using slit-lamp photographs and micro-densitometer and deduced that initial scattering of the lens arises at an
early age in the anterior and posterior outer cortical regions and increases with aging.
Scattering in the nuclear region begins later in life and also increases with aging. Considerable scattering could be detected without significant change in the visual function.
Some authors (Wooten & Geri 1987, Whitaker et al. 1993) have found that stray light,
a functional counterpart of light scatter, did not strongly depend on wavelength. Van den
Berg has studied light scattering and stray light in relation to age (1995), to depth-dependence (1996a, 1997) and to wavelength (1997) in donor lenses. He noticed that the scattering of the superficial layer of the lens was relatively stable and relatively strong compared with that in the nucleus. Only at the age 50 did nuclear light scatter start to increase,
doubling on average by the age of 70. Light scattering in the lens declined strongly with
smaller particle sizes. The small size components dominated for larger than 28 angles,
including backward directions. The particles of significance for forward light scattering
(angles smaller than 28), which is much more important for the patient, were the large
size components (mean radius of 692 nm i.e. on average bigger than the wavelength of
scattered light) and constituted only 0.0003% of the volume fraction of the lens. Van den
Berg has also developed the direct compensation technique for measuring forward light
scatter in vivo (van den Berg & Spekreijse 1987, de Waard et al. 1992, van den Berg
1996b).
The modern noninvasive techniques have given new insights into biochemical changes
occurring during cataractogenesis. When Goldmann (1964) and later Ben-Sira et al.
(1980) used a modified slit lamp image to give a measure of backscatter, has further
developed quasielastic light scattering spectroscopy facilitated quantitative observing the
age-related increase in high molecular weight protein aggregates in the aging human lens
(Tanaka & Benedek 1975, Benedek et al. 1987, Thurston et al. 1997). Nuclear magnetic
resonance spectroscopy offers the ability to probe noninvasively the smaller chemical and
metabolic changes in the lens i.e. changes in motional dynamics (translational and rotational diffusion) of the lens proteins in the cytoplasm level (Datiles III 1992). Laser
Raman spectroscopy monitors aging changes within the lens, so that older nuclear proteins can be easily compared with those newly synthesized in the cortex (Yu & East
1975). It has also been used to demonstrate regional swelling in the outermost fibers of
the lens in diabetes (Datiles III 1992).

20

2.1.2. Classification of lens opacities

There are many subjective ways of classifying cataracts. Various descriptive terms have
been used such as immature / mature, incipient or minimal / moderate or advanced, brunescent / white. According to etiology, cataracts can be divided into age-related, congenital or juvenile, traumatic, associated with intraocular diseases, associated with systemic
diseases and caused by noxious agents such as ionizing radiation or pharmaceuticals.
Cataracts associated with aging are usually categorized into three major, but overlapping,
groups by anatomic location; nuclear, cortical and posterior subcapsular cataracts.
Besides these there are combinations of these three as mixed types of cataract (Datiles III
& Magno 1996).
In an attempt to develop a more uniform, comprehensive classification, photographic
standards to set up various grades of each type of cataract have been published. The
grades are based either on density and color (in the case of nucleus) or according to the
anatomic area of the cataract. Chylack and colleagues have described techniques of standardized photography-based cataract grading. Anatomical description with a system of
cataract photography (Chylack 1978) adopted to the CCRG method (Chylack et al. 1983
and 1984a&b) was designed primarily to be used on isolated lenses in vitro. The LOCS IIII systems (Chylack et al. 1988b, 1989 and 1993) are in vivo systems, with which in
clinical grading the observer may directly compare the patients lens as seen on the slit
lamp with the photographic standard grades. LOCS grading may also be applied to standardized photographs of the lens. Other systems include the Wisconsin system (Klein et
al. 1990) used in the Beaver Dam Eye Study, the Wilmer system used at Johns Hopkins
(Taylor & West 1988, West et al. 1988), the CCESG system used by Sasaki and investigators (Sasaki et al. 1990a) and the Oxford system used by Sparrow with colleagues
(Sparrow et al. 1986, Brown et al. 1987), which was integrated in 1988 technique for
nuclear color matching using Munsell color samples (Sparrow et al. 1988). In this study
the widely used classification systems LOCS II (modified) and LOCS III were used. A
more detailed discussion will appear in chapter 4.2.3.

2.1.3. Quantification of lens opacities

For nuclear documentation conventional slit-lamp photography was found to be less suitable because of the systems limited depth of focus. In the Scheimpflug camera a tilted
film plane and objective maintained the entire slit image in focus (Brown 1969,
Dragomirescu et al. 1978). Scheimpflug images processed with planimetry (Hockwin et
al. 1982), densitometry (Hockwin et al. 1984, Sasaki et al. 1997), nuclear mean density
(Chylack et al. 1988a) and color component subtraction (Kashiwagi & Khu 1990) have
yielded objective measures of the severity of nuclear cataracts. They have also been used
to measure the degree of backscattering from the lens (Sigelman et al. 1974,
Dragomirescu et al. 1978, Hockwin et al. 1982) and the degree of lens autofluorescence
(Brown 1972, Dragomirescu et al. 1978, Lerman et al. 1981). Problems still exist when
the opacity becomes dense and the slit beam cannot penetrate the opacity without becoming diffuse. Also the accurate alignment in both vertical and horizontal meridians is

21
difficult but essential if a series of photographs are going to be compared with each other
(Brown 1972, Dragomirescu et al. 1978).
In retroillumination photographs cortical or posterior subcapsular cataracts appear as
darker shadows against the red reflex. Kawara & Obazawa (1980) were the first to use
polarizing filters on the conventional slit-lamp camera to minimize flash artifacts. Several
image analysis techniques have been introduced including grid counting (Wolfe &
Chylack 1989), thresholding (Kawara et al. 1979, Wolfe & Chylack 1990) and background subtraction (Khu & Kashiwagi 1990). Khu & Kashiwagi also reported a good correlation between subjective classification of cortical and posterior subcapsular cataracts
using LOCS II and corresponding objective measurements by background subtraction.
Also retroillumination photographs can be used to get objective information about the
back scattering properties of the lens (Brown 1971, Maclean & Taylor 1981). As a limitation for the use of retroillumination photographs there is retina based variation of the
color of the red reflex. Processes of the macula, for example diabetic maculopathy, may
whiten the red reflex.
Simultaneous Scheimpflug and retroillumination photography (Sasaki et al. 1987) and
digital video imaging of the lens with combined Scheimpflug and retroillumination optics
(Sasaki et al. 1990b, Sparrow et al. 1990) are also available commercially. They can provide a continuous scale for monitoring lens changes not possible with subjective classification. Adamsons et al. (1991) demonstrated good correlation between clinical grading
and digital analysis of nuclear and cortical opacity.
Scanning photometry (Zeimer & Noth 1984) and other autofluorescence related
methods for measurement of cataracts as well as measurement of back light scatter with
the Interzeag Lens Opacity Meter (Flammer & Bebie 1987, Wegener & Hockwin 1988)
are discussed later.

2.2. History of lens autofluorescence

According to Klang (1948), some years after Stokes fundamental work on fluorescence
(1852) the first investigations about the autofluorescence of the human lens were published by Regnauld (1858) and Setschenow (1859). Since then sporadic reports began to
appear in the literature regarding the nature, origin and function of this phenomenon, but
the results of them were diversified and to some extent contradictory to one another.
Reflected light from the exciting radiation was mixed with the light of fluorescence which
constituted a source of systematic error at the assessment of the intensity and color of the
fluorescence. In determinations of the spectral constitution of the fluorescent light this
source of error has been eliminated by means of observations through selective filters
(Vogt 1913, Merker 1928).
The earliest direct statement on the variation of lens fluorescence intensity with age
may be that of Krienes (1899), who found that the lens fluoresces stronger in elderly
persons. Vogt (1913) found that fluorescence and yellow coloration of the lens both
increased with age and the fluorescence increases with the consistency of the lens, i.e.
with its content of solid constituents. Schanz and Stockhausen published in 19071913
many studies about the fluorescence of the ocular media. They maintained the view

22
previously presented by Widmark (1891 & 1901) and Verhoeff & Bell (1915/16), that the
ultra-violet radiation was injurious to the eyes and responsible for senile cataract among
other affections. They used the fluorescence of the lens as an indicator of the absorption
of the ultraviolet, contributing to spreading knowledge of lens fluorescence and stimulating very many investigations. The demonstration of a reduction of lenticular glutathione
(Adams 1925) was probably the first observation of biochemical alterations in the lens
after exposure to ultraviolet radiation.
Walls & Judd (1933) suggested that the yellow fluorescent pigment in the lens might
serve as an intraocular filter for blue light which is the wavelength of the visible spectrum
subject to the greatest dispersion. Lens fluorescence intensity and dispersion of the fluorescence values were found by Vannas & Wilska (1935) to increase with age. According
to them the intensity of lens fluorescence was about six times as high at 78 years of age as
at 8 years. They also noted exceptionally strong fluorescence in vivo in diabetic patients
with clear lenses. The spectral composition of age-dependent fluorescent changes in the
human lens was first measured clinically by Klang (1942 & 1948). He measured the
intensity of the human lens fluorescence within the spectral regions limited by a blue, a
green and a red filter and was able to demonstrate the presence of at least two different
fluorophors.

2.3. Lens pigments and fluorescence

In the nineteen-seventies more refined chemical and electro-optical techniques enabled
research workers to study autofluorescence, its lifetime and appearance in biological compounds. The identification of spectral changes associated with morphological and biochemical changes were of importance in understanding lens aging. The yellow-brown
coloration of the lens nucleus appeared to be due to fluorescent compounds associated
with the lens proteins, which unfold, cross-link and become insoluble (Pirie 1968,
Zigman 1971, Grover & Zigman 1972, Lerman 1972 & 1976, Kurzel et al. 1973b,
Zigman et al. 1973, van Heyningen 1971 & 1973, Satoh et al. 1973, Dilley & Pirie 1974,
Augusteyn 1975, Spector et al. 1975). At least three mechanisms produce lens pigments
in vitro: photo-oxidation and non-enzymatic glycosylation of lens proteins which are
more widely described, and lipid peroxidation (Borchman et al. 1989).
In intact lenses, Lerman & Borkman (1976) reported an increase in fluorescence
during the first four decades of life without a significant increase in insoluble proteins, the
concentration of which starts rising only during the fifth decade. In lenses with nuclear
cataract they showed a rise in fluorescence some 20 years prior to a rise in the concentration of insoluble proteins. Contrary to that, research on extracts from normal lenses
(Satoh et al. 1973) up to brunescent cataracts (Augusteyn 1975) revealed an increase in
fluorescence with a decrease of water-soluble proteins.

23

2.3.1. Tryptophan
Most proteins are endowed with an intrinsic UV fluorescence because they contain
aromatic amino acids, particularly phenylalanine, tyrosine and tryptophan. Of these three
aromatic amino acids tryptophan has the highest fluorescence quantum yield overshadowing markedly the emissions of the other two. Fluorescence spectra on 3 day and 6 month
old human lenses showed only intrinsic tryptophan fluorescence present (activation
290 nm, emission 332 nm) and no detectable generated fluorogens. Generally one distinguishes between tryptophan and nontryptophan fluorescence. Tryptophan emission
maxima in proteins can vary from 332 nm to 342 nm depending on the protein. Free tryptophan has a characteristic fluorescence emission at 350360 nm. (Lerman 1976)
Metabolism of tryptophan can be presented as follows (van Heyningen 1973): tryptophan => L-N-formylkynurenine => kynurenine => 3-hydroxykynurenine (3HK),
which can react with glucose to O--D-glucoside of 3-hydroxy-L-kynurenine (3HKG) or
break up to 3-hydroxyanthranilic acid and alanine. In physiological concentrations some
of them can act as photosensitizers (Grover & Zigman 1972). Free kynurenine has a blue
fluorescence and is detected consistently only in lenses from the youngest age groups.
3HKG with blue-white fluorescence on electrophoresis paper has no great change with
age and the concentration is similar in the cortex and in the nucleus. Obviously it is
slowly changing into a glucoside closely related to 3HKG but with -amino group absent
or protected.(van Heyningen 1973) More recently (1995) Dillon has reported, that the
concentration of 3HKG actually decreases with age.
The presence of oxygen was found to be an important parameter for determining the
extent to which 3HK reacts with protein. UV-light was not required for activation as it
appeared with kynurenine and 3HKG. However, UV-light was found to increase the
extent of aggregation, pigmentation and development of non-tryptophan AF. The important antioxidant glutathione was found to delay this modification. (Aquilina et al. 1997)
Since lens glutathione concentration decreases with age and is known to be lower in the
lens nucleus than in the cortex (Harding 1970), the lens nucleus appears particularly
vulnerable to oxidative damage. This would also account for the fact that the lens cortex
does not become pigmented with age.

2.3.2. Modification by oxidative damage

Photo-oxidative damage to lens constituents is believed to be an important factor involved
in cataract formation (Spector 1984, Taylor et al. 1988, Dillon et al. 1994, Aquilina et al.
1997). Both intracellular and extracellular oxidative stress affects the lens in vivo. Oxidative damage may adversely affect lens membranes in the cortex as well as proteins in the
lens nucleus. A human lens is well equipped with protective agents against oxidative
stress consisting of superoxide dismutase, catalase and glutathione as well as antioxidant
vitamins such as vitamin C, E and presumably, beta-carotene. Chronic exposure to UVlight and active forms of oxygen may lead to gradual erosion of the antioxidant protective mechanisms and a series of redox reactions with damage of structural proteins,
enzymes and membranes as well as nucleic acids and lipids (Varma et al. 1979, Dillon et

24
al. 1982 & 1985, Jedziniak et al. 1987). Naturally occurring photosensitizers such as Nformylkynurenine and riboflavin may initiate the reaction. Tyrosinase enzyme, which is
present in the human lens, can oxidize one or more of the tyrosine residues in -crystallin
(Lerman 1972).
A significant amount of the lens pigmentation was believed to derive from one or more
aromatic amino acid residues by means of a photo-oxidation process induced by prolonged exposure of the lens to ultraviolet light above 300 nm (Pirie 1971 & 1972, Grover
& Zigman 1972, Kurzel et al. 1973a, Lerman et al. 1976, Dillon 1995). The proposal of
protein tryptophan depletion via oxidation resulting in protein pigmentation (Kurzel) correlates with findings of inverse relationship between tryptophan fluorescence intensity
and extrinsic fluorogen emission intensity in the whole lens (Lerman). Pirie demonstrated that in vitro exposure of lens proteins to sunlight caused yellowing which was
probably the result of photo-oxidation of tryptophan to N-formylkynurenine. Because of
its intrinsic photosensitizing properties, N-formylkynurenine and related species are considered to be biologically very important tryptophan metabolites and oxidation products
(Dillon et al. 1982, Andley & Clark 1989).
While the cornea filters out almost all of the UV light up to 300 nm (Boettner &
Wolter 1962, Ambach et al. 1994), the lens is exposed to the longer UV radiation. The
corneal transmission of light has also been noticed to be quite independent of age (van
den Berg & Tan 1994). Verhoeff & Bell (1915/16), corroborated later by Pitts et al.
(1977), defined the maximum spectral sensitivity of the human lens to be at wavelengths
in 300 nm region. Jose & Yielding (1977) have pointed out that direct damage to lens
DNA may play a role in ultraviolet radiation induced cataract formation.
The two main chromophores that absorb radiant energy transmitted by the cornea in
the young human lens are protein bound tryptophan and 3HKG (Dillon 1995). 3HKG has
its absorption maximum at 365 nm and fluorescence emission at 430 nm. Dillon demonstrated that under continual exposure to 300400 nm UV light, absorption of this light by
3HKG can lead to its photochemical loss with concomitant yellowing of lens proteins. He
proposed that part of this yellowing would be due to the attachment of 3HKG to lens proteins. The yellowing of the lens proteins leads to an increase in the number of photons
absorbed by the lens and UV light may then become a potential causative cofactor in cataractogenesis. Contrary to this, there are many epidemiological studies, which show no
link between cumulative UV-A and -B irradiation and nuclear cataract (Taylor et al. 1988
& 1992, Schein et al. 1994, West et al. 1998). There are even observations, that reduced
growth before one year of age is associated with an increased frequency of age-related
nuclear opacities (Evans et al. 1998).
Lens transmittance below 390 nm is low (Boettner & Wolter 1962, Ambach et al.
1994). Lerman & Borkman (1976) demonstrated that UV light transmission of normal
lenses above 25 years of age was consistently low, about 20% between 300 and 400 nm,
in contrast to the much higher values (7585%) obtained from lenses under 10 years of
age. This was attributed to the relative lack of UV-absorbing chromophores (350400
nm) in the young lens and the increase in these photochemically induced pigments as the
lens ages. Whereas the yellow coloration of the human lens is a consequence of the slow,
natural process of aging, many animals have naturally yellow corneas or lenses for reducing the light scatter of shorter wavelengths (blue light). In fact, a part of blue visible band
(wavelengths shorter than 438 nm) can be cut off without essential effects on the color

25
vision (Aarnisalo 1987). Absorbing blue band up to 480490 nm reduces normal color
discrimination causing a tritan type of color vision defect (Verriest 1963, Aarnisalo
1987). In examination with the Color Vision Meter 712 anomaloscope eyes with the silicone IOL saw more blue than control phakic eyes of patients between 50 to 69 years of
age (Mntyjrvi & Tuppurainen 1996). I found no studies about the actual effects of lens
AF on color vision.
Filtering out light wavelengths shorter than 480 nm, thereby reducing intraocular light
scatter and lens AF, has been reported to improve the visual performance (Zigman 1990).
Some investigators have suggested that increasing short-wave absorption of the lens is
useful in protecting the ageing retina from the blue light hazard (Wolbarsht 1976,
Sliney & Wolbarsht 1980). The human retina, however, can respond to a light stimulus
below 390 nm; both the cone and rod pigments have secondary absorption maxima at 325
and 350 nm, respectively (Dartnell 1957). Thus UV radiation in this region may result in
some visual stimulation and possibly produce color confusion. There is evidence that this
indeed occurs in aphakic patients (Wald 1952, Tan 1971). Wald demonstrated that at 365
nm wavelength an aphakic person could read small letters under circumstances in which
normal observers saw nothing at all. Aphakics saw this region of the spectrum as violet in
color. So acting as an UV protective filter with respect to the underlying vitreous and retina, the lens pigments also have an expedient effect on visual function (Wolbarsht 1976).
It is questionable whether fluorescence excitation studies are truly representative of
absorption by specific fluorescent compounds due to the large amount of scattering, particularly in the cataractous lenses and because quantum yield and self-absorption vary
over the spectrum. Satoh et al. (1973) demonstrated two separate autofluorescence bands
in the human lens. The intensity and the wavelength of the ultraviolet band (maximum at
290 nm for excitation and 333 nm for fluorescence, like tryptophan) was independent of
the age of the lens donor while the blue band (maximum at 348 nm for excitation and 420
nm for fluorescence) increased as a function of age. Spector et al. (1975) obviously presented this same blue fluorophor with excitation maximum at 360 nm and emission at 440
nm.
Lerman & Borkman (1976) described two age-related fluorescent compounds which
develop in the lens nucleus. The first showed activation at 340360 nm with emission at
420440 nm and could be responsible for lens yellowing. It increased with lens age following the same pattern as the increase in insoluble fractions of lens proteins with age
(Clark et al. 1969, Jedziniak et al. 1975). The second, which appeared to be a secondary
product of the former being detectable only after the first decade of life, absorbed light at
415435 nm with emission at 500520 nm. It remained at a relatively low level until the
fourth or fifth decade. A significant increase in the concentration of this fluorogen was
seen in advanced, brown colored nuclear cataracts. Lerman & Borkman also noted a progressive decline in transmission of visible light in the aging lens which could be correlated with the increasing concentration of these pigments in the lens nucleus.
With application of the photoacoustic effect to solid state spectroscopy (Rosencwaig
1973), a new technique became available for obtaining the absorption spectra of amorphous light scattering and opaque samples. Lerman et al. reported in 1978 the results of
their initial studies using photoacoustic spectroscopy (PAS) and in 1981 the results of
densitography measurements. These studies further confirmed their previous results of the
two regions of lens fluorescence.

26
Jacobs and Krohn (1976) found the relative intensities of the fluorescence bands in the
blue-green area of the spectrum shift toward the red as a function of increasing age. This
change was in part attributable to increasing blue absorption and scatter with age but in
part result of a true fluorescence intensity shift. This seemed to imply the existence of two
or more fluorogens within the blue band which change their relative concentration with
age. They found the over-all intensity of the blue-green fluorescence to increase with age
in the same way as the protein bound non-tryptophan fluorescence in studies by Satoh et
al. 1973, Spector et al. 1975, Dillon et al. 1976 and Lerman et al. 1976. They agreed on
the assumption (Zigman 1971, Kurzel et al. 1973b, van Heyningen 1971 & 1973) that fluorogens excited by long-wavelength blue bands were photo-oxidation products of tryptophan, probably kynurenine derivatives. Some other of these fluorogens have been identified as -carboline (Dillon et al. 1976), anthranilic acid and bityrosine (Garcia-Castineiras et al. 1978), whereas others are present but unidentified. As an example Kuck Jr & Yu
(1978) reported of a fluorophor which had a broad emission peak with a maximum of 556
nm when excited at 514 nm.

2.3.3. Glycosylation of lens proteins

In non-enzymatic glycosylation (glycation) glucose binds to amino groups of proteins
leading to formation of advanced glycosylation end-products with the potency to crosslink. There is considerable evidence that non-enzymatic glycosylation of lens proteins is
involved in browning and fluorophore formation in the lens (Monnier & Cerami 1981 &
1982, Liang & Rossi 1990).
The sugar adducts form primarily with the -amino group of lysine residues and they
have been found to increase with age (Garlick et al. 1984). Patrick and colleagues (1990)
suggested that the increase is significant only in early life and that an equilibrium is then
reached between forward and reverse glycation reactions. However, because of the long
half-life of lens proteins, glycation once initiated may progress via a series of reactions
called the Maillard reaction, non-enzymatic browning in the later steps of it. The initial
glycation products can give rise to a complex mixture of products that are pigmented and
can produce cross-links in proteins. They may contribute to the age-related increase in
pigmentation, non-tryptophan fluorescence and non-disulfide covalent cross-links, which
are well-known features of aging lenses, especially brunescent cataracts (Augusteyn
1975, Cerami 1985, Das et al. 1998). Glycosylation end-products absorb light longer than
300 nm and display a strong blue fluorescence (Monnier & Cerami 1981, Liang & Rossi
1990).
Non-enzymatic glycosylation has been shown to occur also with hemoglobin, collagen and nerve proteins (Monnier & Cerami 1982). Glycosylated hemoglobin (HbA1c)
level provides a good estimate of the blood glucose level during the preceding months in
diabetic patients. The corneal autofluorescence (AF) peak which represents the anterior
corneal surface has been considered a combination of scatter, reflection and true AF
(Larsen et al. 1991). An increased AF of the human lens (Helve & Nieminen 1976,) and
the cornea (Stolwijk et al. 1992, Keoleian et al. 1992, Ishiko et al. 1998) has been found
in diabetic patients compared with age-related healthy controls. Corneal AF showed an

27
80% sensitivity and 76% specificity in screening for diabetic retinopathy (Stolwijk et al.
1992).
The lens AF was correlated to glycaemic control and duration of diabetes (Bursell et
al. 1984, Mosier et al. 1986, Occhipinti et al. 1986, Bleeker et al. 1986, Kjer et al. 1987,
Larsen et al. 1989, Lutze & Bresnick 1991, Sparrow et al. 1992). The average extra
decrease of lens transmission index in diabetes has been estimated to 0.5% for each year
of diabetes (van Best et al. 1985b). Even in patients with newly diagnosed diabetes one
has shown higher lens AF values than in their healthy controls (Koefoed Theil et al.
1996). Mori et al. (1997) suggested that lens AF might represent the long-term control of
diabetes and corneal autofluorescence short-term changes in the blood glucose level.
Ishiko et al. (1998) from the same study-group prefer the corneal AF value as indicator of
metabolic control in diabetic patients.
There is a growing awareness of the reductive ability of the lens cell to detoxify the
oxidizing agents and to repair damage, which is based on glucose metabolism. The key
intermediates in the process of either eliminating oxidative agents or repairing oxidative
damage are nicotinamide-adenine dinucleotide (NAD) in its reduced form (NADH) and
nicotinamide-adenine dinucleotide phosphate (NADP) in its reduced form (NADPH). The
use of NADPH in the defense against oxidation is manifested for example by its utilization with glutathione peroxidase in the reduction of oxidized glutathione (Datiles &
Kinoshita 1991). Tocopherols (vitamin E) and carotenoids are lipid-soluble oxidant scavengers that protect biomembranes. Ascorbic acid (vitamin C) and glutathione are important water-soluble antioxidants (Bunce 1991). Ascorbic acid in its different oxidation
states and NADH/NAD redox systems could be in part responsible for the coloring and
the fluorescence of the nucleus (Lohmann et al. 1986, Lohmann 1988). One of precursors of NADH, kynurenine, has fluorescence spectra very similar to lenses with advanced
nuclear cataract. Ortwerth & Olesen (1988) have demonstrated that ascorbic acid or its
oxidation product dehydroascorbate is actually a much more potent glycating agent than
glucose.
In summary, there are two main mechanisms which have been proposed to account for
age-related changes in lens proteins and autofluorescence. One is based on non-enzymatic
glycation and subsequent Maillard or browning reactions between reducing sugars and
amines. The end result of these reactions is the formation of advanced glycolysation endproducts which with cross-linking and insolubilization produce loss of function of proteins. The theory of the second mechanism is based on cumulative oxidative free radical
damage to tissue components. With aging the human lens absorbs an increasing portion of
light from 327 nm to 700 nm (Weale 1988). The selective absorption of blue light is due
to the accumulation in the lens of yellow pigments, some of which are photo-oxidation
products of tryptophan and other products of nonenzymatic glycosylation of lens proteins.
Many of the fluorescent lens pigments are as yet unidentified and their isolation is complicated because they are tightly bound to the insoluble proteins of the lens.

28

2.4. Other recent studies

Hockwin et al. (1984) showed using microdensitometric analysis of Scheimpflug photographs that there is a continuous increase in light scatter during aging in the lens nucleus.
Besides that in the region of deeper anterior cortex increased lens fluorescence was found
starting at 30 or 40 years of age when excited by UV wavelengths of 330380 nm. This
indicates that besides the processes within the nucleus a second process within the lens
cortex is influencing the quality of light transmission in the lens. They assumed that formation of aggregates leading to high molecular weight proteins play a role in the lens
nucleus while in the lens cortex mostly oxidative changes are responsible.
Yappert et al. (1992 & 1993) observed an increase in the specific blue (emission maximum at ca. 450 nm) and green (emission maximum at ca. 520 nm) fluorescence with
increasing age in the soluble and insoluble nuclear fractions. The blue-green fluorescence
in the lens cortex increased toward middle age and thereafter declined or remained relatively constant for older ages. Therefore they assumed that fluorophor formation is not a
marker of aging in the cortical region, as it is in the nuclear region. The blue fluorescence
levels in cataractous fractions were similar to those in clear lens fractions suggesting that
it was not indicator of cataractogenesis. The green fluorescence in the soluble cataractous
fractions showed marked increases compared with the clear lens fractions of similar ages.
This suggests an accelerated formation of the green fluorophores in cataractous lens.
Detailed spectral analyses of the emitted light are rare (Kurzel et al. 1973b, Liang
1987). The collected emitted light consists of a mix of radiations from different fluorophores located in different parts of the lens. This raises problems (Weale 1996a). When
induced by longer wavelengths the emission also shifts to longer wavelengths (Zuclich et
al. 1992). Liang (1990) showed in vitro that the peak emission wavelength of tryptophan
fluorescence increases linearly with age and its intensity predominates over non-tryptophan fluorescence only in young lenses.
Yu et al. 1979 reported the discovery of a red fluorophor in the center of the nucleus of
the older human lens. It had the emission maximum at 672 nm under excitation by 647,1
nm line of a krypton ion laser and its accumulation appeared to parallel the development
of brunescence normally absent before the seventh decade. Their investigations have also
revealed the presence of two other fluorophors; orange 568/591 nm and near-red 568/633
nm. The extrinsic fluorophores indicate changes in the pathologic state of the lens as a
function of age.
Besides AF in the lens and the cornea, the fundus of the human eye also has AF.
Delori (1994) and co-workers developed a noninvasive technique for in vivo measurements of fundus AF using the fundus spectrophotometer. More recently, for imaging and
quantifying the spatial distribution of fundus AF, a confocal laser scanning ophthalmoscope has been used (von Ruckmann et al. 1997a,b & 1998, Solbach et al. 1997). They
found that the fundus AF is derived from lipofuscin at the level of the retinal pigment epithelium. It increases with age. It has been suggested that fundus AF measurements are a
useful non-invasive alternative to fluorescein angiography in identification of eyes at risk
of developing exudative age-related macular degeneration and in the assessment of macular holes. The abnormally high fundus AF is also associated with inherited macular dystrophies, with different fluorophores in different disorders. Moreover laser-excited AF
measurement of NAD(P)H has been applied in studies of mitochondrial function from

29
muscle biopsies in patients with chronic progressive external ophthalmoplegia (Kunz et
al. 1997).
The potential applicability of AF measurement is considerable. It is interesting, that
there are reproducible changes in skin AF with aging. In addition chronic UV exposure
induces the appearance of new fluorophores (Kollias et al. 1998). There are also bluegreen AF in carious human dentine, the intensity of which correlated with the level of
demineralisation (Banerjee & Boyde 1998). The detection of low-quantum-yield AF of
the endogenous coenzyme NAD(P)H is a new tool in probing of biopharmaceutical
effects at the intracellular level (Dellinger et al. 1998).

2.5. Instrumentation development of ocular fluorometry

For fluorometric studies many instruments have been designed to adapt to conventional
slit-lamp microscopes (Langham & Wybar 1954, Maurice 1963, Waltman & Kaufman
1970, Bloom et al. 1976, Brubaker & Coakes 1978). A scanning fluorophotometer, which
measures fluorescence along an axis or in a plane, was described by Zeimer et al. (1983)
and McLaren & Brubaker (1985). In 1988 McLaren & Brubaker described a scanning
ocular spectrofluoro-photometer with the ability to change excitation and emission wavelengths rapidly.
The commercial ocular fluorophotometer, the Fluorotron Master (Coherent Inc., Palo
Alto, California) was presented in 1983 (Zeimer et al. 1983 & 1985, Gray et al. 1985,
Munnerlyn et al. 1985). In 1983 Zeimer et al. compared also two older commercial
fluorophotometers from Gamma Scientific (San Diego) and Metricon (Mountain View,
California) to the Fluorotron Master, which proved to be more appropriate for clinical
research because of its accuracy, reproducibility, improved optics and the availability of
usable computer software.
The International Society of Ocular Fluorometry was founded in Paris in 1982. In
1992 a concerted action on ocular fluorometry stressing standardization and instrumentation development was funded by the European Community (Cunha-Vaz 1993). One of its
main objectives was the development of standardized methods, calibration procedures,
protocols and software for the determination of several ocular parameters using a commercially available fluorometer.
In this study we measured the lens autofluorescence using a scanning fluorometer with
fixed excitation and emission wavelengths of blue-green range, designed, built and now
clinically tested in our department. The schematic representation of the optical system of
the fluorometer and technique of lens fluorometry is presented in chapter 4.2.1.1.

2.6. Analysis of lens fluorometry

Jacobs and Krohn (1981) localized age-related fluorescence within lens sections by scanning microspectrofluorometry. They found anterior and posterior autofluorescence peaks
which were located symmetrically with respect to the lens center, near the transition
between the nucleus and the cortex. The peaks were either equal in height or the posterior

30
peak was higher; a posterior to anterior ratio of 1.17 0.23 (mean SD) could be calculated. Values of 1.08 0.08 (van Best et al. 1985a) and 1.2 0.19 (Zeimer et al. 1987)
have been found by others indicating that the posterior pole showed even 20% more AF
than the anterior one. Larsen & Lund-Andersen (1991) presented a mean value of 1.15 for
the peak ratio found by comparison of scans obtained from either side of the lens.
Since the in vivo profiles usually have anterior peaks that are higher than the corresponding posterior peak, the observed age-related posterior peak extinction has been
attributed to combined light absorption and scattering of the blue excitation and the green
fluorescence. The attenuation of light along the optical pathway involves both excitation
and fluorescence but not necessarily to the same proportion. The phenomenon provides a
clue to the determination of lens transmittance (Zeimer & Noth 1984, van Best et al.
1985a). Larsen & Lund-Andersen introduced in 1991 a mathematical description of the
lens autofluorescence profile and calculations for correcting the distortion in the profile
caused by scatter and absorption of light.
Our own analysis of the lens autofluorescence profile which was undertaken with the
aim of developing a better description of changes in the shape of the autofluorescence
curve during cataractogenesis are presented in chapter 4.2.2.

3. Purpose of the present study

This study was carried out to investigate the blue-green autofluorescence (AF) of the
human lens and to evaluate its clinical applications. The changes of the lens AF profile
with aging and cataractogenesis were evaluated and the results were compared with two
widely used subjective lens opacities classification systems (LOCS II and III). One clinical application that was evaluated more extensively was the relationship between lens AF
measurements and retinal nerve fiber layer visibility in black-and-white images.
Another important parameter concerning lens transmission, the light scatter, was measured using the commercially available Lens Opacity Meter. The scatter values were
compared with the lens AF values and the LOCS grades.
The specific aims of the present study were:
1. To clinically test and to further develop a fluorometer designed and built in our department.
2. To study the influence of aging and cataractogenesis on the blue-green AF of the lens.
3. To study the influence of aging and cataractogenesis on the back scatter of light in the
lens.
4. To determine how the AF and light scatter values are related to each other and how
they reflect the clinical grading of different lens opacities.
5. To evaluate the clinical applicability of various parameters found in the lens AF profile and the sources of error and limitations of our method.

4. Materials and methods

4.1. Patients
4.1.1. Lens autofluorescence and light scatter in healthy individuals
(I and II)
The lens AF and the light scatter were measured from 43 random eyes of 43 healthy volunteers (11 males and 32 females) aged 6 to 86 years, five subjects of each decade. The
subjects had no diabetes or history of other systemic disease. The eyes were required to
be healthy and no significant cataract was allowed. This was ensured with slit-lamp and
direct ophthalmoscopy examinations by the author after the pupil was dilated with two
drops of cyclopentolate hydrochloride (10 mg/ml). Corrected Snellen visual acuity of
better than 0.6 was required except in two subjects over 80 years of age.
In addition, the coefficient of variation (CV) of our fluorometer was tested by measuring the lens AF of ten eyes of ten volunteers ten times. Their ages ranged from 14 to 71
(mean 53.1) years.

4.1.2. Lens autofluorescence and light scatter in cataractous lens

(II and III)
In addition to the 43 healthy volunteers mentioned above, one eye of 84 patients with
varying types and degrees of cataract were examined by the author. The age of the cataract patients ranged from 38 to 93 years with a mean of 70.5 11.9 years. Neither these
subjects had diabetes and the eye had to be otherwise healthy, cataract excluded. This was
ensured by slit-lamp examination and with Volk +78D lens after the pupil was dilated
with two drops of cyclopentolate hydrochloride (10 mg/ml). The definition of cataract is
discussed later. The cataracts were classified into cortical, nuclear, posterior subcapsular
or mixed cataract groups. A subgroup of the healthy volunteers was selected as age
matched controls. A separate control group was selected for patients with posterior sub-

33
capsular cataract as they were younger than the other cataract patients. The age and corrected Snellen visual acuity profile of each of the cataract groups and their controls are
presented in Table 1.
Table 1. Age and visual acuity in each cataract group and their age matched controls.
Patients with posterior subcapsular cataracts were younger and consequently they have
therefore a separate control group.

Cortical
Nuclear
Mixed
Controls

n
25
29
15
13

range
5083
5193
5586
6186

Age (years)
mean SD
71.9 10.0
74.9 10.1
73.3 10.3
72.9 8.1

Posterior subcapsular
Controls

15
15

3871
4168

56.6 9.5
54.2 9.2

Dominating
opacity

Visual acuity
range
0.010.6
0.030.8
0.020.5
0.551.5
0.031.0
0.652.0

4.1.3. Lens autofluorescence and retinal nerve fiber layer evaluation (IV)
Data was collected from 30 randomly chosen eyes of as many subjects with varying
degrees of yellow-brown coloration of the lens in an otherwise healthy eye. The age
ranged from 54 to 77 years (mean 63 6.2 years), corrected visual acuity from 0.6 to 1.2
(mean 0.96 0.11 ) and intraocular pressure from 10 to 23 mmHg. No diabetic patients
were included. The eyes were examined with slit-lamp and Volk +78D lens by the author
after dilatation of the pupil.

4.1.4. Lens autofluorescence, light scatter and LOCS III (V)

We examined both eyes of all the 141 men aged 57 to 76 years (mean 64 4.9 year), who
participated in projected eye examination associated in the U.S. National Cancer Institute
supported alpha-tocopherol, beta carotene lung cancer prevention study (The ATBC Cancer
Prevention Study Group 1994 a & b) between 22-26 February 1993 in Helsinki. The participants were male smokers from 50 to 69 years of age at entry in 1984-1985. They were
recruited from the total male population of this age group in 14 geographic areas in southwestern Finland and randomly assigned to one of four supplementation regimens: alphatocopherol (50 mg per day) alone, beta carotene (20 mg per day) alone, alpha-tocopherol and
beta carotene or placebo. Follow-up had been continuing for 5 to 8 years in February 1993.
Primarily all pseudophakic and aphakic eyes were excluded as well as 4 subjects with
diabetes, as diabetes affects the lens AF properties. There were no cases of eye trauma
included, based on the eye history questionnaire. One subject with corneal dystrophy was
excluded because of disturbance of light transmission through the cornea. For the statistical analysis the data of the left eye of all subjects (n=122) was selected. After biomicros-

34
copy the pupils were dilated and definition of lens opacities as well as AF and light scatter measurements were performed as described next.

4.2. Methods
4.2.1. Description of the instrumentation
4.2.1.1. Fluorometry
The autofluorescence of the lens was measured using a fluorometer designed and built by
Heikki Nieminen and, during these investigations, clinically tested and further developed
in our department. Fig. 1. is a schematic presentation of the optical system of our fluorometer as well as its analyzing and recording setup.
Light source
Condensing lens
Filter
A and B

Slit

Mirror

Scanning motor

Ocular and
Filter D

Slit
Field lens

Eye

Filter C

Signal amplifiers

A/D D/A

PC-microcomputer

Laser printer

Photodiode

Oscilloscope
V/F
converter

Tape
recorder

F/V
converter

Plotter

Fig. 1. Schematic presentation of the optical system of our fluorometer and its old and new
analysing and recording set-up seen from the side. Filter A is an infrared absorption filter.
Excitation filter B and filter D have peak transmission at 495 nm and barrier filter C has peak
transmission at 520 nm.

35
The excitation light source is a 25 W incandescent lamp from Zeiss biomicroscope fed
by a current-regulated power supply. Infrared light is blocked by an absorption filter (A).
The wavelength of excitation light is set by an interference filter (B) with peak transmission at 495 nm. A barrier interference filter (C) has broader peak transmission from 520
nm. Transmission characteristics of these filters are shown in Fig. 2. It is important to
exclude any transmission of reflected or scattered excitation light through the barrier filter
by minimizing the overlap between filters. The author uses term blue-green autofluorescence to describe exited and emitted light. In fact, the AF which is measured is green.

Fig. 2. Transmission characteristics of the infrared absorption (left), and the excitation and
barrier filters (right) used for scanning fluorometry.

The optical system consists of a moving motorized lens and a fixed lens that focus the
excitation light on the eye and collect emitted as well as back scattered light from the eye.
The dimensions of the illumination slit measured at the focal plane in air are 0.1 x 1.0
mm. The beam is scanned linearly along the optical axis of the eye at an angle of 20
while the subject views a fixation target. Fluorescence emitted from the lens passes the
lens system on the opposite side at a symmetrical angle and is transmitted through a slit
and a barrier filter to a photodiode detector (EG & G Electro-Optics: UV-040 BG, spectral range 250 nm1150 nm). Any blue light due to scatter within the eye is reflected by
the barrier filter and focused in front of the ocular. The instrument is calibrated before
each measurement using a fluorescent reference surface. Since the instrument is mounted
on a slitlamp-like table, it is easily positioned so that the focal plane seen as blue through
the ocular is centered in the pupil of the patients eye and just inside the corneal reflex. A
weak corneal AF peak can also be measured, if the scan is started in the front of the cornea. Each unidirectional scan from the anterior chamber to the vitreous takes about 3 sec
and was monitored at first with an oscilloscope. Later on the scan was instantly visualized
on the screen of the PC-microcomputer. False measurements (eye movements, eyelid
blinks) can be immediately rejected and the eye rescanned before analysis and storage of
the data.
No contact lens was used. The long-term stability curves of the entire fluorometer
showed good stability at two different fluorescence levels (I: Fig. 3). The AF values as a

36
function of the distance in the eye were processed by low-noise amplifiers and signals
were visualized with an oscilloscope, plotted on paper and via V/F converter stored on
tape (Fig. 1.). All the analysis and measurements from the AF curve were at first made
from the paper printout. After the first article we continued the development of the
device. We incorporated another blue interference filter (D) with peak transmission at 495
nm in front of the ocular to reduce leaks of light from the examination room. From the
fourth article the AF curve was digitized with a A/D converter and processed by PCmicrocomputer with a specific software written by Heikki Nieminen. The PC-microcomputer controlled also the calibration and the scanning system. The graphic fluorescence
profile could be shown on the computer screen and with a PC-mouse one could choose
the desired measuring points. After the calculations the results were printed on paper with
or without the graphic scan and stored in the table form of Microsoft Excel. The original
digitized AF curve was stored in the other file.

4.2.1.2. Lens Opacity Meter

In articles II, III and V the back light scattering from the lens was measured with the commercially available Lens Opacity Meter 701 (Interzeag AG, Schlieren-Zurich, Switzerland). First experiences and description of the device have been reported by Flammer &
Bebie (1987) and Wegener & Hockwin (1988). The device measures the light scattered
back from a modulated light beam ( = 700 nm) of 1,5 mm in diameter which is directed
along the optical axis into the eye. The patient looks at a green fixation target in the
instrument or, when prevented by poor visual acuity, the instrument is aligned with the
centre of the dilated pupil. A minimal pupil size of 4 mm or more is recommended
(Clarke et al. 1990, Yao et al. 1992). This could usually be achieved by two drops of
cyclopentolate hydrochloride (10 mg/ml). A portion of the back scattered light is detected
by a sensor at a fixed angle of 27 below the incident beam, processed by a built-in computer and displayed as a numeric value on an arbitrary scale between 0 (clear lens) and 99
(dense cataract). The examination is very quick and reproducible. A built-in computer
controls automatic calibration, storage of measurements, rejection of false measurements
(eyelid blinks), and data analysis. 250 readings are taken with one push of a button in 0.5
seconds and electronically stored as a single (averaged) measurement. Five such consecutive measurements were taken for each eye. A printed paper strip gives the values of each
measurement, mean value and standard deviation. Statistical analyses were performed on
the mean value as it appeared on the printout of the device.

4.2.2. Our analysis of lens fluorometry

The device produces a graphic fluorescence profile of the lens which consists of anterior
and posterior juxtacortical peaks and a central plateau (Fig. 3). All the measurements
from the AF profile of each case in these five articles were done by one examiner (the
author). The lens fluorescence maxima originate from a layer near the transitional zone
between the cortex and the nucleus, not from the surface of the lens. (Jacobs & Krohn

37
1981) The height of the anterior peak (Grays peak; Mosier et al. 1983) is used as a measure of the maximum AF value. The lens surfaces are located at the half-peak-height
intercepts of the anterior and posterior slopes of the lens AF curve (Zeimer & Noth 1984,
Larsen et al. 1991).
Zeimer & Noth (1984) and van Best et al. (1985a) assumed that the maximum AF is
approximately the same in the anterior and posterior parts of the lens since the lens is
rather symmetrical in structure. Any difference in AF intensity between anterior and posterior parts can then be attributed to a loss of exciting and fluorescent light in the lens by
scatter and by absorption in the nucleus and cortex. Because of the small difference in
peak wavelength between exciting and barrier filters the average transmission index (T)
for blue-green light was calculated according to T = Fp Fa where Fa and Fp is the
value of the peak AF in the anterior and posterior part of the lens, respectively. They also
pointed out that values obtained with this equation apply to blue-green light only; the
transmission index for white light will be somewhat higher. As a result from this equation a certain transmission value can occur with a low as well as with a high average lens
AF value.
In the first article maximum lens AF and light transmission index were analyzed as a
function of age. Since it appeared that transmission index could not be determined in
advanced cataracts due to the posterior peak extinction and the AF profiles differed not
only in height, but also in width between the cataract groups (Fig. 6), the width of the AF
curve at height of 50% (W50) and 75% (W75) of the anterior peak was determined in
article III. Several width to maximum AF ratios were calculated in order to describe the
shape of the AF curve. In the next articles (IV and V) newly developed software allowed
us to measure areas of the AF curve. The area of the AF curve above half height of the
anterior peak (A50) was measured. Total area was not used in order to avoid disturbance
of scattered light near both lens surfaces (tailing; Mosier et al. 1983, Occhipinti et al.
1986). The ratio A50 / maximum AF was compared with other AF parameters to describe
changes in the lens AF profile.
The changes in the axial AF profile are markedly affected by attenuation of light in the
lens. Inspired by Larsen (1993) the loss of the maximum lens AF value caused by scatter
and absorption in the lens was corrected for and the ratio maximum AF / transmission
index was evaluated previously unpublished. High absorption in the lens, that is a low
transmission index, may obviously mask a high intrinsic lens AF (Larsen 1993: Fig. 13).
Thus division by the transmission index may potentially be used to correct the measurements of maximum AF to estimate the lens AF as it would appear on a scan where each
layer of the lens was successively removed as the scan proceeds from the anterior to the
posterior layers of the lens. However, the posterior AF peak and thus the transmission
index proved to be difficult to define exactly from a lens with advanced cataract also in
article V.

4.2.3. Evaluation of lens opacities

In the first article the lens was evaluated by slit-lamp examination with the pupil dilated
and considering the age, the eyes with significant cataracts were excluded.

38
In following articles (II to IV) the lens opacities observed in the slit-lamp examination
were evaluated using colored photographic standards modified from the Lens Opacities
Classification System II (LOCS II, Chylack et al. 1989). The slit beam was angled
through both sides of the dilated pupil so that the entire lens could be examined. The posterior subcapsular opacities were graded in retroillumination. The number and length of
cortical spokes, both opalescence and colour of the nucleus, and the width of posterior
subcapsular opacities were graded from 0 for clear lens to 3 or 4 for advanced cataract. In
the original LOCS II there is only three (NC 0-2) grading categories for nuclear colour
and six (C 0-5) grading categories for cortical opacities. In articles II and III the cataract
eyes were classified into cortical, nuclear or posterior subcapsular cataract groups or
mixed opacities groups when two or three cataract types were significantly presented
(grades 2 to 4) at the same time. All these lens gradings were done by one examiner (the
author).
In article V we used the improved lens opacities classification system, LOCS III
(Chylack et al. 1993). With its expanded sets of reference photographs (six slit-lamp
images for grading nuclear color and nuclear opalescence, five retroillumination images
for grading cortical cataract and five retroillumination images for grading posterior subcapsular cataract) and decimalized grading, LOCS III provides a more sensitive grading
than LOCS II when applied to photographic images of cataracts. With 0.1-unit intervals,
nuclear color (NC) and nuclear opalescence (NO) scores summarizes the nuclear color or
opacity of the lens in one numeric dimension between 0.1 (colorless or clear) to 6.9 (brunescent or very opaque). In cases of cortical and posterior subcapsular cataract the scale
ranges from 0.1 to 5.9.
Two Neitz (Kowa Optimed Inc., Torrance, California) retroillumination CT-R blackand-white photographs with Kodak Tri-X 400 film were taken from each eye after pupil
dilatation, one focused on the anterior surface and one on the posterior surface of the lens.
One Topcon 6M slit-lamp (Tokyo, Japan) color photograph was also taken using a slit
width of 0,2 mm, a slit beam orientation of 45, focused on the center of the nucleus and
using Kodak Ektachrome ASA 100 HC slide film. The films were numbered and filed for
later grading performed with the LOCS III by the Center for Clinical Cataract Research,
Boston.

4.2.4. Evaluation of retinal nerve fiber layer

Retinal nerve fiber layer photographs were taken by Heikki Nieminen with a Canon CF60 ZA wide angle camera (Canon Inc., Kawasaki City, Japan) using the same blue interference filter of 495 nm wavelength as used for AF excitation. Kodak Tmax-100 low-sensitivity, high resolution black-and-white film was used. The camera was aligned and the
fundus focused using the cameras built-in side marks and split-lines focusing devices.
The detailed technique of RNFL photography has been described earlier (Airaksinen &
Nieminen 1985).
RNFL photographs were evaluated in a completely masked fashion by Juhani
Airaksinen with no clinical information available. The RNFL visibility scores ranged
from 1 for very poor to 5 for very good visibility of the nerve fibers. The intraphoto-

39
graphic error of this type of RNFL scoring has a coefficient of variation of 15%
(Airaksinen et al. 1984).

4.2.5. Statistical methods

The statistical analysis of the data was performed using the SOLO software package
(BMDP Statistical Software Inc., Los Angeles, California) in articles I to IV and the software SPSS 6.1.2. for Windows (SPSS Inc., Chicago, Illinois) in articles IV and V.
Mean, standard deviation (SD) and range are given for most of the continuous variables. The coefficient of variation (CV) of our device was calculated for the maximum
AF and the transmission index by dividing the square root of the mean of the ten
variances by the average of the ten mean values of the ten eyes.
In articles I and II both linear and curved relationships between maximum AF, transmission index, light scatter and age were analyzed using multiple regression techniques
(Armitage 1980). Two-tailed unpaired Students t-test was used to compare the mean
values of age, maximum autofluorescence and light scatter between different cataract
groups. Analysis of variance (ANOVA) and Kendalls rank correlation were used in
analysis within cataract groups. (II and III)
In article IV linear correlation coefficients (Pearson) were calculated between the RNFL
visibility score and different AF parameters as well as visual acuity. A further analysis using
regression technique was made to find out whether quadratic relationship between RNFL
visibility score and various AF parameters would improve the model. To evaluate the
variability of the RNFL visibility score by lens AF parameters and age, a multiple regression analysis by the forward stepwise method was performed with RNFL visibility score as
the dependent variable and AF parameters and age as the independent variables.
To evaluate the relationship between the various LOCS III cataract grading parameters
and values of different lens AF parameters, light scatter and age, a multiple regression
analysis with the various LOCS III parameters alternating as the dependent variable were
performed. Both linear and quadratic methods were used. A forward stepwise procedure
was used to find the order of significance of AF parameters, light scatter and age when
LOCS III grades of nuclear color and nuclear opacity alternated as the dependent variable.
The data of only one eye of each individual was included in the study, because the correlation between eyes in the same individual violates the assumption of statistical independence among all sampled eyes. The level of statistical significance was set at p < 0.05
in every case.

4.2.6. Ethical approval

The protocol for the study was approved by the Ethical Committee of the Medical Faculty, University of Oulu. The study followed the tenets of the Declaration of Helsinki.
Informed consent was obtained from all participants after the nature of the study had been
explained.

5. Results
5.1. Lens autofluorescence and light scatter in healthy individuals
Typical AF profiles for lenses of different ages are shown in Fig. 3. Both peak height
difference (transmission index) and maximum AF increase with age. The coefficient of
variation was 3.9% for the maximum AF and 2.9% for the lens transmission index.

Fig. 3. Typical autofluorescence curves of healthy individuals aged 7 years (A), 53 years (B) and
84 years (C). Maximum autofluorescence increases and lens transmission index decreases with
age. Note different scale in A.

Among these healthy people aged from 6 to 86 years the maximum AF ranged from 1.2
to 95.0, the light scatter value from 8.0 to 40.8 and the computational transmission index
value from 1.0 to 0.79. The maximum AF and the light scatter were both highly significantly related to age. The best fit was obtained with an exponential regression model (r =
0.95; p<0.0001 and r = 0.94; p<0.0001, respectively). In Fig. 4 the regression line of maximum AF started to increase in early childhood from about zero value whereas a little light
scatter was always present in the lens. The coefficient of determination (r2) indicates that
about 90% of the total variation in the maximum AF as well as the light scatter values could
be attributed to age. As it could be expected from Fig. 4, there was also a positive correlation between maximum AF and light scatter measurements (r = 0.90; p<0.0001).

41

Fig. 4 A. Regression of maximum autofluorescence with age. The data can be best fitted by the
equation: maximum AF = 0.00952 + 0.55184 x age + 0.00674 x age2 with a coefficient of determination r2 of 0.91.

Fig. 4 B. Regression of light scatter values with age. The data is best fitted by the equation: light
scatter = 9.4234 0.0239 x age + 0.0037 x age2 with a coefficient of determination r2 of 0.88.

42
The lens transmission index (T) for blue-green light as a function of age is presented in
Fig. 5. Lens transmission is almost unchanging or merely decreases very slowly with age
up to 60 years. Thereafter lens transmission decreases rapidly and the interindividual variation increases. The data is best fitted by the equation (solid line in Fig. 5) T = 0.98270
0.304 x 108 x age4 (r = 0.82; p<0.0001).

Fig. 5. Regression of lens transmission index (T) with age.

Both maximum AF and light scatter values were equally correlated to the lens transmission index (r = 0.80; p<0.0001).

5.2. Lens autofluorescence and light scatter in cataractous lenses

Age and visual acuity profiles of various cataract groups and their controls are presented
in Table 1 (chapter 4.1.2). The mean visual acuities in the cortical and mixed cataract
groups were statistically significantly lower than those of the nuclear and posterior subcapsular cataract groups (p<0.0001).
Typical AF profiles from lenses of healthy controls as well as cortical and nuclear cataracts are shown in Fig. 6. The mean value of the maximum AF as well as the light scatter
in each cataract group differed statistically significantly from that of age matched healthy
controls (Table 2).

43

Fig. 6. Typical AF profiles from lenses of (A) healthy control, (B) cortical and (C) nuclear
cataract.

Table 2. Maximum autofluorescence and light scatter values (mean SD) in 84 eyes with
different types of cataract and their age matched controls. Patients with posterior
subcapsular cataracts were younger and consequently they have therefore a separate
control group.
Dominating opacity

Maximum AF

Cortical

25

58.34 25.03

86.69 24.68

Light scatter

Nuclear

29

120.54 32.93

45.46 13.23

Mixed

15

98.45 20.75

53.48 21.29

Controls

13

78.65 16.08*

28.00 5.54*

Post.subcapsular

15

73.79 31.70

43.61 15.86

Controls

15

46.15 14.11**

19.01 5.74*

* p < 0.0001; ** p < 0.01

The highest AF values were measured in nuclear cataracts where the maximum AF was
also related to an increasing yellow-brown colour of the nucleus (Kendalls tau = 0.48;
p<0.01) (III: Table 3). In cortical cataracts the maximum AF was significantly lower than in
the control eyes. The mean maximum AF values between nuclear and mixed type cataracts
as well as between cortical and posterior subcapsular cataracts did not differ statistically significantly from each other (Table 2). Posterior peak extinction (Fig. 6) was also found statistically significantly (Chi2 = 11.69; p<0.01) more frequently in cortical cataracts (23 of 25
eyes; 92%) than in eyes with other types of cataract (32 of 59 eyes; 54%). Therefore, the

44
transmission index could not be determined in all cataractous eyes. The lowest determinable
transmission index values were between 0.70 and 0.80 and corresponding Snellen visual
acuity between 0.2 and 0.5.
The highest light scatter values were measured in the cortical cataracts. The mean scatter values of the nuclear, mixed and posterior subcapsular cataract groups did not differ
statistically significantly from each other (Table 2). There was a weak but statistically significant correlation between scatter values and cataract grades in nuclear cataract
(Kendalls tau = 0.28; p<0.05), but not in cortical and posterior subcapsular cataracts.

Fig. 7. Scatter plot of light scatter values and corrected visual acuity in 84 cataractous eyes with
corresponding regression line.

In the total group of 84 cataractous eyes, the light scatter was statistically significantly
correlated to visual acuity (r = 0.71; p<0.0001, Fig. 7) whereas the maximum AF had no
significant correlation to visual acuity or to light scatter. However, there was a large
variation in visual acuity with light scatter value below 60 and the achieved good correlation was obviously basically due to those 17 observations in the upper corner of the figure. Also in the various cataract subgroups a statistically significant correlation between
scatter values and visual acuity could be found with the highest correlation in cortical
cataract (Table 3). Only in the nuclear cataract group was the correlation between maximum AF values and visual acuity statistically significant.

45
Table 3. Relationship between maximum AF as well as light scatter values and visual
acuity in 84 eyes with cortical, nuclear, posterior subcapsular and mixed cataract; linear
regression.
Opacity

Maximum AF
n

Light scatter

Cortical

25

0.26

NS

0.79

< 0.0001

Nuclear

29

0.60

0.0005

0.47

< 0.01

Post.subcapsular

15

0.42

NS

0.63

< 0.02

Mixed

15

0.05

NS

0.74

< 0.002

The most applicable width/maximum AF ratios to describe differences in the shape of

AF curves between various cataract groups are presented in Table 4. W50 and W75 represent the width of the AF curve at a height of 50% and 75% of the anterior AF peak. In the
nuclear and mixed cataract groups the ratio W75 to Fa (maximum AF) was statistically
significantly lower than in other groups (p<0.0001), which characterise the high and narrow AF curve of the nuclear cataracts and most AF curves of mixed cataract group. The
ratio (W50W75) to Fa was statistically significantly higher in cortical cataracts than in
the other groups (p<0.0001) indicating that the AF curve is low and flattened in cortical
cataract. This ratio makes it possible to discern them from the other cataract groups as
well as from healthy eyes of the younger age. However, there was no correlation between
LOCS III cortical grades and this ratio in article V.
Table 4. Mean values SD of the two ratios which best described the qualitative
differences in shape of AF curve between various cataract groups. W50 and W75
represent the width of the AF curve at height of 50% and 75% of anterior AF peak and Fa
is the height of anterior AF peak, in other words maximum AF value.
Opacity

W75 / Fa

Cortical

0.22 0.16

(W50W75) / Fa
0.15 0.12 *

Nuclear

0.10 0.06 *

0.06 0.03

Post.subcapsular

0.28 0.17

0.08 0.03

Mixed

0.11 0.05 *

0.07 0.02

Controls

0.22 0.09

0.07 0.03

* p<0.0001

46

5.3. Lens autofluorescence and retinal nerve fiber layer evaluation

Variation of the lens fluorometric data and visual acuity is presented in Table 5. An
example of RNFL photographs and corresponding lens AF curves for excellent and poor
visibility of the retinal nerve fiber bundles are shown in Fig. 8. Correlation coefficients of
RNFL visibility to lens AF parameters and to visual acuity are presented in Table 6.
Table 5. Range and mean SD for the lens fluorometric data and visual acuity of the
study population (n = 30). W75 is the width of the AF curve at a height of 75% and A50 is
the area of the AF curve above the half height of the anterior peak.
Range
Maximum AF (Fa)

45 207

Mean SD

102 49

Transmission index (T)

0.68 1.0

0.90 0.08

W75 / Fa

0.14 0.49

0.34 0.11

A50 / Fa

0.13 0.84

0.42 0.20

Maximum AF / T
Visual acuity

45 285

118 68

0.60 1.20

0.96 0.11

47

Fig. 8 A. An RNFL photograph and the corresponding lens AF curve for excellent visibility of
the retinal nerve fiber bundles (RNFL visibility score 5, maximum AF (Fa) 45, transmission
index 1.0, A50/Fa 0.84 and visual acuity 1.2).

48

Fig. 8 B. An RNFL photograph and the corresponding lens AF curve for poor visibility of the
retinal nerve fiber bundles. Note different scale on y-axis. (RNFL visibility score 2, maximum
AF (Fa) 207, transmission index 0.84, A50/Fa 0.15 and visual acuity 0.6).

49
Table 6. Pearson correlation coefficient (r) and statistical significance (p) between RNFL
visibility score and lens autofluorescence (AF) parameters or Snellen visual acuity (n = 30).

Maximum AF (Fa)

0.58

0.0008

Transmission index (T)

0.42

0.020

W75 / Fa

0.41

0.023

A50 / Fa

0.48

0.007

0.59

0.0007

0.39

0.031

Maximum AF / T
Visual acuity

The relationship between RNFL visibility and the ratio between the maximum AF and
the transmission index showed the highest linear correlation (r = 0.59; p = 0.0007). A
pure maximum AF showed nearly equal linear correlation (r = 0.58; p = 0.0008, note
misprint in the original paper) which could be slightly improved by using a quadratic
regression instead of a linear model (Fig. 9). The coefficient of determination (r2) was
0.35 for both AF parameters which means that more than one third of the total variation in
RNFL visibility data could be attributed to lens AF.

Fig. 9. Scatter plot of RNFL visibility score and maximum autofluorescence with corresponding
regression line. The data is best fitted by the equation: y = 3.925 + 0.0021 x 7.131 105
x2 with a coefficient of determination (r2) of 0.35.

50
Regression analysis using the RNFL visibility score as the dependent variable indicated that the lens AF measurements provided a better prediction of the RNFL score than
did the patients age. In the stepwise regression model only the ratio maximum AF to
transmission index was included but age and other AF parameters were excluded because
they did not improve the model i.e. did not reduce the residual standard deviation statistically significantly (p = 0.15).

5.4. Lens autofluorescence, light scatter and LOCS III

Variation of the lens fluorometric data, the light scatter values of the Interzeag Lensmeter
and the LOCS III grades for the different cataract types are presented in Table 7. In 4
cases of 122 (3%) the height of the posterior lens AF peak for transmission index could
not be reliably define.
Table 7. Range and mean SD for the lens fluorometric data, the values of the Interzeag
Lensmeter and the LOCS III grades for the cataract types of the left eyes of 122 subjects
included the study.
Range

Mean SD

Maximum lens AF (Fa)

36 186

78.5 26.3

Transmission index (T)*

0.70 0.99

0.89 0.06

A50 / Fa

0.15 1.13

0.50 0.20

36 226

87.9 33.0

15.8 73.8

27.7 8.27

Cortical cataract

0.1 3.6

0.49 0.73

Nuclear color

1.9 6.3

2.73 0.73

Nuclear opalescence

0.4 5.2

1.41 0.93

Posterior subcapsular cataract

0.1 4.1

0.21 0.53

Fa / T*
Interzeag Lensmeter
LOCS III

* n = 118
The relationship between the nuclear color and the lens transmission index showed the
highest linear correlation (r = 0.71; p<0.0001). The coefficient of determination (r2) was
0.50, suggesting that about half of the total variation of the transmission index values was
accounted for by the nuclear color. Scatter plots with corresponding regression lines of
nuclear color and (A) maximum AF, (B) transmission index, (C) light scatter and (D) age
are showed in Fig. 10.
The results of the regression analysis using the various LOCS III parameters alternating as the dependent variable are shown in Table 8. The regression analysis using nuclear
color as the dependent variable indicated that the transmission index provided a more precise prediction about the nuclear colour than age did. The residual standard deviation of
the regression model including age alone was 0.21 larger than that having transmission
index alone as the independent variable (0.63 and 0.42, respectively).

51
Table 8. The regression of LOCS III grades of cortical cataract, nuclear color, nuclear
opalescence and posterior subcapsular cataract, respectively, with different lens AF
parameters, light scatter and age.
LOCS III grades

Maximum
AF (Fa)
(n = 122

Transmission A50/Fa
Fa / T
Light
index (T)
scatter
(n = 118)
(n = 122) (n = 118) (n = 122)

Age
(n = 122)

NS

NS

NS

NS

0.30*

0.31*

0.67**

0.71**

0.67**

0.60**

0.68**

0.54**

Nuclear opalescence

0.55**

0.51**

0.49**

0.42**

0.64**

0.39**

Posterior subcapsular
cataract

NS

NS

NS

NS

NS

NS

Cortical cataract
Nuclear color

* p<0.005; ** p<0.0001

In a stepwise multiple regression analysis adding age to the model including transmission index alone as the independent variable, reduced the residual standard deviation only
by 0.02 (from 0.42 to 0.40; p<0.005). Adding other AF parameters or light scatter values
to the regression model including transmission index alone did not add statistical significance to the model.

52
A

Fig. 10 A & B. Scatter plots with corresponding regression lines of nuclear color and
(A) maximum AF and (B) transmission index.

53
C

Fig. 10 C & D. Scatter plots with corresponding regression lines of nuclear color and
(C) light scatter and (D) age.

54
The light scatter had a stronger correlation to nuclear opalescence (r = 0.64; p<0.0001)
than AF parameters or age did (Table 8). However, among these 118 cases with determinable transmission index value, in the stepwise regression analysis with nuclear opalescence as the dependent variable only the transmission index was entered to the model.
Adding light scatter value, other AF parameters or age to the regression model including
transmission index alone did not improve the model statistically significantly. Values of
maximum AF and light scatter as well as LOCS III grades of these four cases with undeterminable transmission index are presented in Table 9. Obviously these values clearly
improved single correlation values of light scatter and maximum AF to nuclear opalescence.
Table 9. Values of maximum AF and light scatter as well as LOCS III grades of the four
cases with undeterminable transmission index.
Case 1

Case 2

Case 3

Case 4

99

121

186

153

27.6

56.0

73.8

61.6

Cortical cataract

0.2

0.7

1.2

3.6

Nuclear color

4.9

5.3

6.3

4.1

Nuclear opalescence

1.7

5.2

4.7

4.5

Posterior subcapsular cataract

0.1

0.1

0.1

0.2

Maximum AF
Light scatter
LOCS III grades

As it was expected, there was no correlation between AF measurements and the grades
of cortical or posterior subcapsular cataract. A weak but statistically significant correlation could be found between the grades of cortical cataract and light scatter values as well
as age (Table 8).
The light scatter was statistically significantly related to the maximum AF (r = 0.54;
p<0.0001). That was about the same size as obtained in the nuclear cataract group (r =
0.61; p = 0.0004) in article III. The correlation between the light scatter and the transmission index was also on the same level (r = 0.54; p<0.0001).
Although a certain transmission value can occur with a low as well as with a high average lens AF value (4.2.2. and Fig. 6), relationship between the transmission index and the
maximum AF values was statistically significant (r = 0.68; p<0.0001, Figure 11).

55

Fig. 11. Scatter plot with corresponding regression line of transmission index and maximum
AF.

6. Discussion
6.1. Lens autofluorescence and light scatter in healthy individuals
The maximum value of blue-green AF in the lens and back scatter of light measured with
the Interzeag Lens Opacity Meter (LOM) increased with age in a very similar fashion in
healthy eyes (Fig. 4.). So far there have been no studies to compare the LOM and AF
measurements.
Our results showed a statistically highly significant correlation between age and lens
AF in healthy, non-diabetic individuals. According to the regression line AF started to
increase in early childhood from about zero value. Elderly people in the 8th decade had
almost 4 times greater maximum blue-green AF in the lens compared to individuals in the
3rd decade. This is in agreement with previous studies (Jacobs & Krohn 1981, Zeimer &
Noth 1984, Bursell et al. 1984, Occhipinti et al. 1986 and van Wirdum et al. 1988). Theil
et al. have found more recently (1996) about 3.4 x increase in lens AF of healthy subjects
between these decades. In most studies the rate of age-related AF change has been linearly calculated. In our study, however, the coefficient of determination was improved
statistically significantly, when a quadratic function of age was added into the model.
The amount of large insoluble lens protein molecules increases with age and specific
fluorogens are associated with this protein fraction (Lerman & Borkman 1976). Since
scatter of light increases with the volume of the molecule (Philipson 1969) these large
protein molecules have also a greater ability to scatter light than the small molecules. We
found a statistically highly significant correlation between the scatter values and age in
non-cataractous eyes. An age related increase in light scatter of the human lens has been
previously reported by Sigelman et al. (1974), Ben-Sira et al. (1980), De Natale et al.
(1988) and recently van den Berg (1995), among others. Ben-Sira measured the image
luminance of the backscattered beam. He noted a gradual increase in light scatter values
for the anterior cortex from the age of 20 years, whereas scatter values for the nuclear
region did not begin to increase until after 50 years of age. That is well in harmony with
the quadratic relationship between light scatter and age in our study. De Natale &
Flammer (1992) reported also a quadratic correlation between LOM values and age in an
unselected population.

57
Van Best et al. (1985a) obtained approximately the same fluorescence values in the
anterior and posterior juxtacortical regions when scanning human donor lenses from both
sides. The posterior peak reduction obviously represents optical changes due to absorption and dissemination of light in the aging lens. This provides, with certain limitations, a
clue to the determination of lens transmittance. The advantages of measuring the transmission with the use of the AF peak ratio are its simplicity and the fact that it is part of the
data obtained while measuring AF. Therefore, it does not necessitate a special measurement. A potential alternative to this could be the measurement of lens transmission from
colour transparencies (Seland et al. 1992). The authors concluded however, that the photographic pigments in the transparencies did not reliably mimic the transmission characteristics of human lens pigments. Also a mathematical pattern has been presented to estimate lens transmission from the LOCS III nuclear colour score (van den Berg & Felius
1995).
It is clearly discernible from figure 5, how little the lens transmission for blue-green
light, evaluated from the AF curve, changes during the first 60 years. One can conclude,
that age-related changes in lens AF reflect parallel changes in the yellow coloration of the
lens. These changes, however, do not necessarily impair the ability of the lens to form a
good retinal image. Thus a black-and-white grating seen through a yellow lens may obviously appear black-and-yellow to the aged observer, but the pattern as such may be
equally as visible as it is to the younger observer. On the other hand, the lens absorption
and back scatter of light have been reported to increase with age (Said & Weale 1959,
Mellerio 1971, Sample et al. 1988). Boettner & Wolter (1962) showed a very little
change of direct transmittance of the lens in blue-green range between lenses of 4 year
old and 53 year old subject, whereas Lerman & Borkman (1976) found marked decrease
already between 8 year old and 25 year old lenses.
After the age of 60 the transmission of blue-green light seems rapidly to decrease and
the interindividual variation increases, which is in concert with other published data (van
Best et al. 1985a, Occhipinti et al. 1986, Mosier et al. 1986, van Best & Kuppens 1996).
This is in agreement also with the observation of Ben-Sira (1980) and van den Berg
(1995) who found a significant increase of nuclear light scatter after the age of 50. Also in
our LOCS III study light scatter had a significant correlation with the transmission index
among subjects at the age of over 60 in average.
A certain transmission value can occur with a low as well as with a high average lens
AF value. The observed correlation between AF and transmission values could be in part
due to the relationship of both phenomena with age. It could also indicate that the substances that cause absorption are also connected to fluorescence emission, as suspected by
others (Lerman & Borkman 1976, Occhipinti et al. 1986). This is in agreement with our
findings that the transmission index provides a more precise prediction to the LOCS III
nuclear color grade than age did. The nuclear and perinuclear cortical regions of the lens
show the most intense blue-green autofluorescence, (Jacobs & Krohn 1981) which may
be derived from a UV-light induced tryptophan photodegradation reaction or by nonenzymatic glycosylation of the lens proteins.
Recently van Best et al. (1998) reported quantitative changes in maximum AF and
transmission index of the lens between 1983 and 1996 in a group of healthy subjects.
They found a mean yearly increase of lens AF of 8.69 unit, which was 35% larger
increase than that calculated on the basis of age-dependence curves in 1983. Transmission

58
index showed a mean yearly decrease of 0.508%, which was 130% larger decrease than
expected. They supposed that these changes could be attributed to the increased exposure
to solar UV radiation in that period of time and agreed with the hypothesis about light
affecting the lens (Lerman & Borkman 1976, Weale 1996b). Then again, in epidemiological studies long-term exposure to sunlight (UV A and B and visible radiation) has turned
out to be a possible risk factor for cortical opacification, but no association with either
nuclear opacity or nuclear colour has been found (Taylor et al. 1988 & 1992, Schein et al.
1994, West et al. 1998).

6.2. Lens autofluorescence in cataractous lenses

Cataract interferes with the retinal image by scattering and absorbing light as well as by
increasing the lens autofluorescence. In cortical cataract, there is an imbalance in electrolytes leading to overhydration of the lens and to disruption of the lens fibers. This is followed by an irregular protein distribution in the lens and fluctuation in the refractive
index which increases scatter (Philipson 1973, Duncan et al. 1989 & 1997). In the nuclear
region, there are increases in selective spectral absorption, in pigmented chromophores
and in cross-linked protein aggregates (Philipson 1973, Augusteyn 1975, Weale 1985). It
has been proposed that the decreased transmission of visible light in the aging lens and in
nuclear cataract is mainly due to the accumulation of lens fluorogens rather than configurational changes of the lens proteins as seen in cortical cataracts (Lerman & Borkman
1976).
When the cataractous lens is assessed with the help of the slitlamp, the difference
between a nuclear and a cortical cataract is obvious. This difference in morphology is also
well expressed in our lens AF measurements. The cataractous nucleus has more marked
AF than the cortical cataract. This is in agreement with the biochemical analysis of the
cataractous lens. The fluorescent proteins are mainly found in the nuclear region of the
lens (Spector et al. 1975, Liang et al. 1988, Das et al. 1998). Nuclear fractions of the lens
show a steady increase in blue and green fluorophores with age. In the cortical fractions
there is no increase in blue-green fluorophores after 40 years of age indicating that
beyond this age, AF is not a marker of aging in this region (Lerman & Borkman 1976,
Yappert et al. 1992). The LOCS III provides an expanded and more sensitive scale for
both nuclear color and opalescence grading.
An increase in green fluorescence (emission maximum at ca. 520 nm) of the lens was
postulated to be a precursor of cataract (Lerman & Borkman 1976, Mosier et al. 1986,
van Wirdum et al. 1988, Yappert et al. 1992) whereas the blue fluorescence (emission
maximum at ca. 450 nm) levels in cataractous and noncataractous lenses were similar.
Cortical and nuclear cataracts with their totally different etiology and morphological
appearance may both arise from oxidative mechanisms; one taking place primarily at the
surface membranes and the other within the nuclear proteins. This may help explain why
the majority of senile cataracts are, in fact, mixed in form. (Duncan et al. 1997)
With maximum AF measurements we could separate every cataract group from age
matched healthy controls (Table 2.). Maximum AF was most distinctive in nuclear and
mixed cataract groups and was related to increasing nuclear colour. It was somewhat

59
unexpected that the maximum AF did not have a stronger relation to LOCS III nuclear
colour grades. Instead, the highest correlation coefficient was found with the transmission
index.
The mean AF value in cortical cataracts was even lower than in the age matched
healthy controls (Table 2). This may be attributable to combined light absorption and
scatter in the blue-green band. Obviously cortical opacities prevent excitation and thereby
emitted light from passing through the lens. Lerman & Borkman (1976) proposed that the
anterior cortical opacities block the UV light from penetrating the nucleus and prevent
them from generating free radicals which may induce tryptophan photodegradation. Yu et
al. (1979) discovered a red fluorophor in the nucleus with emission maximum at 672 nm
under excitation by the 647.1 nm line of a krypton ion laser. The lens cortex is more
transparent to these wavelengths, so that the fluorescence of the nucleus may be measured
without marked interference from cortical absorption. Red fluorescence was measurable
in the normal human lens at the age of 70 and it was found to parallel the development of
nuclear brunescence.
The transmission index could not be calculated in every case. The height of the posterior lens AF peak could not be exactly determined in advanced cortical or nuclear cataracts due to the posterior peak extinction. The only evidence of its presence might be a
bend on the posterior slope of the lens AF curve. That causes an additional fluctuation to
transmission index values below 0.80. Van den Brom et al. (1990) and Larsen & LundAndersen (1991) have reported about the same problem. They presented a mathematical
correction for distorted AF profiles caused by attenuation of light along the scanning
pathway in the lens.
The lowest determinable transmission index values in our study were 0.70 (III and V)
with corresponding Snellen visual acuity about 0.2 (III). Obviously in the cases with
transmittance below 0.70, due to strong absorption of light, AF cannot be measured from
the whole lens thickness but only from the anterior part of it. That is why the estimation
of the whole lens transmittance by means of blue-green AF measurement is not possible
in such dense cataracts. It may be noted that in our LOCS III study, there were 4 of 122
patients whose transmission indexes could not be determined. These individuals also
showed the highest defined values for cortical and for both nuclear LOCS III grades
(Table 7 & 9) as well as the highest measured values of the maximum AF and the light
scatter (Case 3 in Table 9).
We introduced two width-height ratios to describe the changes in the shape of the AF
curve which occur in various types of cataract during cataractogenesis. The ratio W75 to
Fa (maximum AF) seemed to be sensitive to increasing nuclear cataract. The other ratio
(W50 W75) to Fa was indicative of the flattened AF profiles in cortical cataracts. However, this ratio had no correlation with LOCS III cortical grades. The small cataractous
changes in the shape of the AF curve were explicitly manifested in the top part of the AF
profile. That is why the area based ratio (A50 / Fa) was developed being more sensitive to
those small changes in the AF curve than the width based ratios. After all, if in dense cataracts AF is measured only from the anterior part of the lens, the value of the anterior lens
AF peak, that is maximum AF, seems to be the most reasonable parameter. Also this
parameter is attenuated due to absorption, the effect is larger in lenses with low transmittance.

60
The relationship between AF and nuclear color, nuclear opacity as well as visual acuity in nuclear cataracts imply that AF measurements might be used as a rapid and objective way of obtaining a quantitative estimate of the amount of nuclear colour and transmission. There is a connection between the nuclear colour and nuclear opalescence as
they both increase with age. Their LOCS III grades were highly correlated with each
other (r = 0.57; p<0.001). The exceptionally low AF values in cortical cataracts yield
information about the loss of light in the lens cortex, but a significant correlation between
AF measurements and the grades of cortical cataract could not be found.

6.3. Light scatter in cataractous lenses

The influence of scatter on the total transmittance is complicated. Although both absorption and back scatter of light increase with age and with the formation of cataract, scatter
is thought to be less dependent on wavelength than absorption (Wooten & Geri 1987,
Whitaker et al. 1993). Elsewhere the scatter has been expected to be greater at lower
wavelengths (Jacobs & Krohn 1976, Seland et al. 1992). With age, the scatterers in the
lens increase in size, therefore the wavelength dependence of scattering may change
(van den Berg & Felius 1995). Boettner and Wolter (1962) measured direct and total
transmittance in an attempt to separate the scattering effects from pure absorption. The
direct transmittance was much lower than the total transmittance, especially at short
wavelengths, which indicates a pronounced influence of scattering.
Reduction of the retinal contrast is caused by an increase in forward scatter, which
cannot be directly derived from the backscatter (Elliott & Hurst 1989, de Waard et al.
1992). However, a good correlation between the Lens Opacity Meter (LOM) readings and
the improvement in the postoperative visual field was found for nuclear and also for cortical cataracts (de Natale & Flammer 1989, Yao & Flammer 1993) whereas in their well
executed and careful study Crichton et al. (1990) did not find any correlation. A relationship between LOM values and contrast sensitivity has also been found (Rouhiainen et al.
1996). The LOCS system as well as the Lens Opacity Meter, both used in this study, measure the back scatter of light.
In article II, a subgroup of cortical cataract included 25 eyes, 17 of which had a highly
advanced, nearly white cortical cataract with very poor visual acuity (Fig. 7.). Their LOM
values exceeded the scale and were marked 101 in the results. The relative lack of variation in this subgroup causes bias and some error to the results, which one has to take into
consideration.
The mean LOM value of each cataract group differed statistically significantly from
that of age matched healthy controls (Table 2.) with the highest values in cortical cataracts. Elliott & Hurst (1989) as well as Lee & Taylor (1990) found LOM values not useful
in documenting the presence or severity of cortical cataract due to a considerable variation of the readings. Jones & Kratz (1990) and de Waard et al. (1992) found paradoxically
very low LOM values with marked cortical opacities and poor visual acuity.
We found the highest correlation between LOM values and visual acuity in cortical
cataract in agreement with Stoltenberg et al. (1989). However, the association between
LOM values and LOCS III cortical cataract grades was very weak. Poor performance of

61
the opacity meter in cortical cataracts was detected by others (Costagliola et al. 1990,
Strobel et al. 1990, Bonomi et al. 1990). It has been explained with the limitations of the
1.5 mm diameter LED beam, which measures a cylindrical volume of the lens along the
optical axis. Because the cortical region may contain not only heterogeneous scatter
zones, but they may be located in optically peripheral positions, the Lensmeter is suitable
mainly for quantifying homogenous opacities (Wegener & Hockwin 1988). In our study
(II) cortical spokes extended as far as to the optical zone at least in one sector of the lens
in every eye among the cortical cataract subgroup. Due to the operation principle the
opacity meter is less suitable for studying asymmetric, local or slight anterior or posterior
subcapsular cataracts.
Suitability of the opacity meter for identifying different types of cataract is further
stressed by the fact that while cataract groups could be separated from the healthy controls, only the cortical cataract group could be separated from the other types of cataract
(II). However, with the photographic grading (LOCS III) the relationship between LOM
values and nuclear opacity and even nuclear color was much more significant than relationship between LOM values and grades of cortical cataract. Although the LOM values
had superiority to AF parameters in association with nuclear opacity, in the stepwise
regression analysis, among these 118 cases with determinable transmission index value,
adding the LOM value to the transmission index value did not improve the prediction of
nuclear opacity or colour.
The nuclear scatter zones develop relatively homogeneously across the optical center
of the lens and could therefore be better detected. The more distinct the observed cataract,
the more variation was found in light scatter values within each cataract group (II: Fig. 2).
This is consistent with the increased variability on test-retest values for advanced cataracts found by Sample et al. (1991). A great degree of overlap between the severity of
nuclear opacity and LOM values found by Lee & Taylor (1990) indicates the poor specificity of this device. Due to large interindividual differences, the mean for a group cannot
be applied to individual patients.
Our results with healthy individuals show that the lens can produce considerable light
scatter and AF without a significant reduction in visual acuity. Besides, there was a large
variation in the Snellen visual acuity with light scatter values below 60 in cataractous
eyes (Fig. 7). This may indicate that light scatter measurements, at wavelength as long as
700 nm are not sensitive to cataract-induced changes in visual acuity or maybe traditional
Snellen visual acuity is deficient in quantifying cataract (Assia et al. 1997). Testing glare
and contrast sensitivity might be more useful.
Posterior subcapsular cataract cannot be estimated by either AF or light scatter measurements. The results of this study suggest, that the Lens Opacity Meter does not add
significantly to the information obtainable with LOCS III and fluorometry.

6.4. Clinical applications

There is a definitive need for a simple and cost-effective objective technique to quantify
lens opacities. Such a method would be useful for instance in epidemiological studies on
risk factors and preventive strategies for cataracts. In epidemiological studies different

62
anatomical types of lens opacities should be distinguished, because each type may have
unique biochemical properties and may probably be initiated by different factors (Duncan
et al. 1997). It is also important to detect true change in cataract status beyond variation
due to measurement error, grader error or other noise in the assessment. A widely used
subjective technique was developed by Chylack and associates. They designed the Lens
Opacities Classification System, LOCS I (Chylack et al. 1988b), which was further developed to LOCS II (Chylack et al. 1989) and LOCS III (Chylack et al. 1993). The LOCS
III, with its expanded sets of reference photographs and decimalized grading, provides a
more sensitive grading than LOCS II when applied to photographic images of cataracts.
The lens fluorogens are responsible, to a significant degree, for the increasing yellow
coloration of the lens with age (Augusteyn 1975; Lerman & Borkman 1976). The
observed color of the lens depends, among other things, on absorption and backscatter of
light. In grading the nuclear opalescence by a slit-lamp examination the nuclear colour
can cause over- or underestimation of the lens opacity grade. Among the three main types
of cataract, the grading of nuclear cataract is considered the most problematic (Sasaki et
al. 1997).
We have developed a technique to measure the blue-green AF of the lens and we have
shown that it increases with age and with the degree of nuclear cataract. The lens AF has
been proposed to be visually important at excitation wavelengths of 420 nm and less
(van den Berg 1993). However, the lens AF measurements can also be used for evaluating
other lens transmission properties such as scattering and absorption of light (Zeimer &
Noth 1984, van Best et al. 1985a). There was a large variation in the transmission for
blue-green light in older individuals within the same age group.

6.4.1. In evaluation of retinal nerve fiber layer

In glaucoma RNFL evaluation may provide information on early and minimal loss of
optic nerve axons that cannot be detected by other techniques (Airaksinen & Heijl 1983).
Confounding factors in the clinical evaluation of RNFL photographs are subjectivity,
technical variations in photographic quality, variation of pupil size and fundus pigmentation. The visibility of the RNFL decreases with age (Jonas & Schiro 1992). An agerelated loss in the transparency of optic media may be the cause. In healthy eyes also the
actual number of axon bundles has been shown to decrease with age (Mikelberg et al.
1989). However, the effect of the loss of approximately 4000-5000 axons per year (0.4%
of the total optic nerve axon count) on the visibility of the RNFL has not been determined. Moreover, the normal axonal number variability is greater than its decline with
age (Repka & Quigley 1989, Jonas et al. 1992).
In RNFL photography short wave-length blue light is commonly used to obtain good
quality images. The blue light is beneficial as it is reflected back to the camera from the
superficial layers of the retina (the RNFL) and does not penetrate into the deeper layers as
longer wavelength light does. In eyes with yellow-brown nucleus the blue light is blocked
by the yellow lens and cannot penetrate into the eye. Various attempts have been made to
measure and classify the colour of the human lens, but none has received universal acceptance. This results from problems of poor reproducibility or difficulty with sophisticated

63
technical procedures. The Topcon SL-45 Scheimpflug camera with black-and-white or
colour film (Dragomirescu et al. 1978, Khu & Kashiwagi 1993) provides quantitative
measures of nuclear opacification but not of the lens colour. Subjective grading of the
lens with a slit-lamp and colour photographic standards (West et al. 1988, Sparrow et al.
1988, Chylack et al. 1993) may be the most widely utilised assessment technique. In this
study we showed that nuclear colour and opacity correlated well with our lens AF measurements. Therefore, we used the lens AF to express the amount of lens yellowing. This
enables us to take mathematically into account the amount of decreased image quality
caused by the yellow-brown nuclear colour of the lens.
Generalized loss of retinal nerve fibers without localized abnormalities is the most
common mode of development and progression of early glaucomatous abnormalities
(Tuulonen & Airaksinen 1991). It is also much more difficult to detect than localized
damage. We were able to demonstrate how lens yellowing in our study expressed as the
lens AF - had an actual effect on RNFL visibility in black-and-white images taken with a
monochromatic filter. All the lens AF parameters reflecting nuclear opalescence and
colour were superior to Snellen visual acuity in predicting RNFL scoring in our sample.
In a clinical situation when evaluating RNFL photographs knowledge of the amount of
lens autofluorescence will make the judgement easier: high AF and low RNFL visibility
might indicate normal RNFL after all, while low AF and poor visibility of the RNFL may
imply true RNFL thinning - on the supposition that the patient has no cortical or posterior
subcapsular cataract. Glaucoma or ocular hypertension have no significant effect on lens
AF and transmission (van Best & Kuppens 1996).
The maximum AF value seemed to be very useful. It can be defined without difficulty. However, it increases not only with increasing nuclear opalescence but also with
age as early as in childhood (Fig. 4A). Because the age had not been fixed in our study, it
acts as a confounding factor. However, adding age to the multiple regression analysis
model including only the maximum AF value as the independent variable did not reduce
the residual standard deviation statistically significantly. No doubt, there was a large variation of RNFL scores for a given AF value but the variation decreased clearly in higher
AF values (Fig. 9).
The shape of the AF profile remains quite constant for a long period of time in healthy
eyes although the level of AF increases (Fig. 3). Because the transmission index and other
parameters describing the shape of the AF curve are less affected by age, they may therefore reflect the changes in transmission properties of the lens better than the maximum
AF value itself. The posterior AF peak and thus the transmission index is difficult to
define exactly from a lens with advanced cataract. However, the quality of RNFL photographs in such case is so poor, that cataract surgery could be recommended to facilitate
the follow up. The width-height ratio (W75/Fa) is not sensitive to small cataractous
changes in the shape of the AF curve, since these explicitly manifest themselves in the top
part of the AF profile. For this reason the area based ratio (A50/Fa) had a better correlation with the RNFL visibility score.
There are several factors that influence the visibility and the clinical evaluation of the
RNFL in fundus images. Among such factors are the age-related changes in RNFL visibility. These changes are related on one hand to the actual loss of axon bundles, and on
the other hand to age-related changes in lens colour and the maximum AF value, caused
by changes in the molecular structure of the lens. Furthermore, there is an increasing

64
variation of lens transmission properties with age over 60 years. These cataractous
changes are best described with the lens transmission index value. Therefore, it might be
useful to combine these parameters when analysing the transmission and AF properties of
the lens as a confounding factor in clinical RNFL evaluation.
In the future the assessment of the optical disturbances using lens AF measurements
may be a helpful additional tool in identifying true RNFL thinning. The ratio of the maximum AF to the transmission index is in a way a transmission corrected maximum AF
value. A lower transmission index gives a higher corrected maximum AF value and takes
into account the attenuation of light in the lens. This ratio had the highest linear correlation with the RNFL visibility in our study. Further examination with a larger sample size
is needed for showing the potential superiority over a pure maximum AF value.

6.4.2. In correction of perimetry results

For providing a full characterization of glaucomatous damage the structural and functional measures are both important. Cataract may affect the visual field before any change
in central visual acuity is evident (Klewin & Radius 1986). Fig. 4A shows how blue light
induced AF increases with age. Thus, as an individual ages, he or she also has less blue
light reaching the retina. The transmission of blue light is more dependent on the amount
of lens pigments than the white light and it can vary considerably above the age of 50,
depending on the lens coloration of each individual (Johnson et al.1988). It is clearly seen
also in our study how this variability of lens transmission properties remains even if individuals would be age matched (Fig. 5). The average age matched transmission values of
the lens reported in the literature will not serve well for individual cases in older age
classes. That is why in blue-on-yellow perimetry an automated method of specifying the
colour and the light transmission properties of the lens would be of particular interest as a
means of normalizing or establishing a common baseline among different patients undergoing this test.
There is a need to separate optical attenuation from that caused by neural attenuation.
The reduced transmission caused by lens opacities will lead to difficulty in the detection
of diffuse loss of nerve fibers and an underestimation of the depth and / or area of any
focal visual field loss. Because nuclear cataract has been shown to produce similar sensitivity reductions for blue-on-yellow and white-on-white perimetry (Moss et al. 1995),
such a comprehensive normative data base, which accounts for the degree of light scatter
and absorption for a given patient, would be useful for correcting the effects of optical
attenuation in white-on-white perimetric profile also. Elegant but time-consuming techniques have been designed to correct for the spectrally selective light losses in the lens
(Sample et al. 1988, Johnson et al. 1989, van den Berg & Felius 1995).
In view of the above, we were wondering whether it would be possible to obtain a
good estimate of lens transmission with AF measurement. We found a statistically highly
significant linear correlation of blue-on-yellow mean sensitivity to lens transmission
index (Teesalu et al. 1995). It is also proposed, that the reference level for correcting
blue-on-yellow perimetry results could be determined more precisely using fluorometry
(transmission index) of the lens than with age alone (Teesalu et al. 1997). However, the

65
variability was further decreased when also age was added to the model. Because maximum AF was highly significantly correlated to age, use of transmission corrected AF
value (maximum AF / transmission index) alone might be sufficient for correcting the
effects of optical attenuation and ageing, particularly if 440 nm blue light is used for excitation. It is worth reminding, that the second fluorophor of Lerman & Borkman (1976)
with blue-green AF expressly absorbed light at 415-435 nm.
There are limitations for correcting perimetry results with fluorometry. Transmission
index value cannot be calculated in every case. Obviously when the lens has very low
transmittance, AF cannot be measured from the whole lens thickness but only from the
anterior part of it. That is why the estimation of the whole lens transmittance by means of
AF measurement is not possible in such dense cataracts. Also cortical opacities interfere
with excitation and thereby emitted light from passing through the lens and AF can seem
to be even lower than in the age matched healthy controls. Posterior subcapsular cataract
may decrease the lens transmittance and affect the perimetry results without showing in
the lens AF curve.

6.4.3. In epidemiological studies

Quantification of lens opacities is essential in clinical trials of medications or dietary supplementations, like the ATBC Cancer Prevention Study, which we were associated with
in one of our studies (V). The intake of riboflavin, vitamin C, vitamin E and beta carotene
has been found to reduce the risk of cortical, nuclear and mixed cataract types (Leske et
al. 1991). However, supplementation with alpha-tocopherol or beta carotene for 5 to 8
years did not influence the cataract prevalence in the ATBC Cancer Prevention Study
(Teikari et al. 1997).
For longitudinal studies grading techniques should be reproducible, easy to use and
sensitive enough to distinguish true changes in the lens status. The lens AF measurement
is an objective and reproducible, noninvasive method providing information on the transmission properties in the central region of the lens. The good correlation with the nuclear
grades of the improved and widely used LOCS III classification system confirms that fluorometry may be a useful additional tool together with a subjective grading system in the
follow-up of optical changes occurring in the nuclear region of the lens.

6.4.4. In the future

There are some improvements for the device itself as well as the software connected,
which might be reasonable to pursue before further studies. However, the intraindividual
variability of our AF measurements (CV 3.9% for maximum AF and 2.9% for the lens
transmission index) is good and our results are fully comparable to those achieved with
the more expensive commercial ocular fluorophotometer (Bleeker et al. 1986, van Best &
Kuppens 1996). Besides the maximum AF value, which is significantly age-dependent,
the shape of the AF curve reflects the transmission properties of the lens and is less linearly affected by age (Fig. 5). The usefulness of combining both parameters, for example

66
the ratio of maximum AF to the transmission index, would need further evaluation. Such
a ratio might take into account age-related physiologic changes as well as the lens transmission properties when acting as the lens descriptive parameter in clinical tests.
The co-operation with the International Society of Ocular Fluorometry as well as with
other closely connected researchers would be beneficial. The increase in exposure to solar
UV radiation in the North due to the thinning of the ozone layer and its theoretical potential for causing lens damage should motivate further AF studies. Measurements of corneal
and lens AF would be of particular interest, because of their objectivity, reproducibility
and clear association with morphological and biochemical changes in these tissues.
Photo-oxidative damage can be shown in vitro in lens membranes in the cortex as well as
in proteins in the lens nucleus. Because these proteins are replaced naturally very slowly,
they are prone to accumulate damage from environmental and intrinsic effects. However,
the recent epidemiological studies have not found association between cumulative UV-B
radiation and nuclear cataract (West et al. 1998).

6.5. Improvement topics: Reduction of sources of error and limitations

of the method
Our technique used for the measurement of lens AF was highly reproducible. However, it
involves sources of error, some of which are referable to deficiencies of the device and
some have their origin in the heterogeneous properties of the eye and the lens.
We used as the excitation light source a 25 W incandescent lamp. During the aging of
the lamp the intensity of its radiation diminishes and its spectral composition may change.
Fluctuations in voltage will also result in different levels of brightness and spectral variations. These may affect the measurements, because AF intensity is practically always proportional to the intensity of the exciting radiation. According to Elenbaas (1972) only 5 to
10% of the total radiation emitted by an incandescent lamp is within the visible spectrum
(400 to 780 nm), whereas 80 to 95% of the energy consists of infrared radiation (wavelength longer than 780 nm). We incorporated the infrared absorption filter in our fluorometer to block leaks in longer wavelengths which might cause additional error. No significant drift occurred in the measurements of two different fluorescence levels over a thirtyminute period (I: Fig. 3).
Calibration of the fluorometer is essential to obtain reliable lens AF measurements and
it corrects partly for the alteration of an ageing incandescent lamp. We used a commercially available fluorescent reference surface before each measurement. A fluorescein
solution of known concentration and constant pH is also recommended (Boets et al.
1992), particularly for long term measurements. We measured the relative AF and did not
study the amount of actual AF in each individual lenses. The specifications of our device
are thus somewhat wanting as we did not compared the relative units with fluorescein
testing solutions of two or more known concentrations in order to check the linearity of
the instrument. That would have enabled us to convert readings to units of concentration
of fluorescein and made it easier to compare results with those obtained by others.
It is essential to eliminate any transmission of reflected or scattered blue light through
the barrier filter leading to pseudofluorescence. We used high quality interference filters

67
for setting the wavelengths of excited and emitted light. The overlap of their transmission
bands is minimal as shown in Fig. 2. Munnerlyn et al. (1985) recommend to increase the
separation between the excitation and barrier filters to about 30 nm at the 50% transmission points and possibly doubling both filters for maximum reduction in the overlap.
Our fluorometer was designed to reduce the depth of focus in order to achieve the best
longitudinal resolution. This can be obtained by both reducing the slit width and increasing the angle between the exciting and the emitted beam which determines the depth of
the focal diamond (Munnerlyn et al. 1985). Then again, any reduction in the slit width
or increase in the angle reduces the available signal, thus increasing the minimum detectable signal or increasing the time required to make a measurement. An exact analysis of
the axial resolution has yet to be made. It is a shortcoming of the device, but its effect on
the results is negligible. It is important to know the axial resolution in vitreous fluorophotometry, when the high concentration of injected fluorescein in the choroid may influence the vitreous value. We are measuring lens AF without any artificial fluorescence
injection and the lens is situated far from possible fundus AF. The development of our
device is now temporarily stopped because of the death of Mr Heikki Nieminen, who was
mostly responsible for the design, construction and all improvements of this fluorometer.
The means to improve our fluorometer might be the use of a better excitation source.
A quartz halogen lamp has increased energy in the blue spectral region since it operates at
a higher temperature than conventional bulbs. More expensive, but also a better way
would be to use a laser, e.g. an argon laser with a wavelength selector as the excitation
module (Mc Laren & Brubaker 1985). This would also make it easier to reduce the leakage through the barrier filter and to improve the longitudinal resolution, because the laser
beam is relatively monochromatic and coherent. At the low light levels used in ocular fluorophotometry, photon-counting detection systems, in which each photon represents a
single digital pulse, can be used to effectively reduce noise due to current leakage in a
photomultiplier (Munnerlyn et al. 1985). This means of detection would also be very useful in a system such as our latest version, in which all of the signals are digitized and processed by a computer.
We measured the lens AF only from the slit area scanned along the optical axis of the
eye. Cycloplegia was used to reduce accommodative artefacts during the lens scan.
Although in the lens nucleus fluorophores are quite homogeneously concentrated in the
center (van den Berg 1993), other regions of the lens tend to be more variable. Consequently lenses with locally denser cataractous areas, as it frequently appears in cortical
cataracts, will cause large measurement variation. Dense cortical opacities may almost
totally prevent excitation and thereby emitted light from passing through the lens. Posterior subcapsular cataracts, although commonly centrally located with clear consequences
for visual performance, cannot be quantified either with AF measurement or with the
commercial Lens Opacity Meter. Because the anterior and posterior lens AF peaks are
located near the transition between the nucleus and the cortex, posterior subcapsular cataract does not come out with transmission index calculations, either (Table 7 & 9). In these
cases the retroillumination photographs with objective measurements using background
subtraction probably give the best results (Khu & Kashiwagi 1990).
There are variations in the spectral composition of the lens fluorescence. The corneal
irregularities and corneal AF as well as secondary fluorescence from scattered blue light
in the eye (Gray et al. 1985) tend to obscure in vivo measurements. The image blurring

68
commonly called tailing is inherent to the instrument and unique to each measured eye.
Moreover, the fundus of human eye has also actual AF derived from lipofuscin (Delori
1994, Solbach et al. 1997).
The lens transmission index was calculated according to equation, which assumes that
the posterior and anterior lens AF peaks are equal in height. However, the posterior peak
is in fact somewhat higher than the anterior peak (see chapter 2.6). By the comparison of
AF scans obtained from either side of the isolated human lens Larsen & Lund-Andersen
(1991) found a peak ratio Fp/Fa of 1.15 in average. Inserting this value into the equation
T = Fp / Fa (Zeimer & Noth 1984) we come to a paradoxical value 1.07 of for the maximum transmission. For practical reasons 1.00 was chosen in the present study to represent
the value of maximum transmission. Thus, the transmission values achieved in this study
for blue-green light may be slightly higher than those determined by more direct methods.
The height of the posterior peak could not be exactly determined in advanced cataracts due to the posterior peak extinction. In spite of the improved analysis software, the
fluctuation of measured transmission index values increases below the 0.80 value and
below 0.70 the transmission index is undeterminable. The relative ease, however, with
which data may be obtained makes the transmission parameter suitable, in most cases, for
comparative analyses in the ageing and cataractous lens. Zeimer & Noth (1984) used
peak-tangent extrapolation estimates for definition of the anterior and posterior AF peaks,
but the reproducibility of the transmission measurement by this method was only 11.7%.
We decided to use uncorrected values, because they are more reproducible. Additionally,
we attempted to improve the software and reduce the noise for better accuracy also when
measuring transmission in moderate cataracts.
It is an evident limitation that our fluorometer is used with only one fixed excitation
and emission wavelength pair. For excitation Heikki Nieminen selected the same blue
interference filter of 495 nm wavelength which was used for RNFL photographs taken on
black-and-white film. An appropriate filter is an essential factor in RNFL photography
and Ducrey et al. (1979) reported the best visibility of nerve fibers at 495 nm for human
eyes with clear optic media. Heikki Nieminen noticed how an increasing yellow coloration of the lens nucleus together with the blue filter influence the quality of RNFL photographs. One of the purposes of this study was to investigate whether the AF measurements could be used for correcting this effect when evaluating RNFL photographs. On the
other hand, the flexible use of several fixed excitation and emission wavelengths from
400 nm to 700 nm might allow measurements of a large number of potentially useful fluorophores. The red AF would be measurable without marked interference from cortical
absorption. Possibility for stepping through a range of wavelengths would allow the measurement of the excitation and emission spectrum of a fluorophore. For correcting blueon-yellow perimetry results, lens fluorometry with 440 nm excitation, which is the stimulus wavelength of blue on yellow perimetry, might improve the result over the 495 nm
excitation used in the study of Teesalu et al. (1997).

7. Summary and conclusions

Age-related and cataractous changes in the human lens interfere with retinal imaging by
causing scatter and absorption of light as well as autofluorescence (AF), particularly at
short wavelengths. The results of many tests used in ophthalmic diagnosis and in vision
research depend on the amount of light reaching the retina. If we could correct for the loss
of light in the lens of a given individual, the data would be improved. There is a need for
an objective and reproducible method to quantify lens opacities also in epidemiological
studies on risk factors and preventive strategies for cataracts.
It has been proposed that the decreased transmission of visible light in the ageing lens
and in nuclear cataract is mainly due to the accumulation of lens fluorogens rather than
configurational changes of the lens proteins as seen in cortical cataracts. The lens fluorogens are responsible, to a significant degree, for the increasing yellow colour of the lens
with age. The nuclear and perinuclear cortical regions of the lens show the most intense
blue-green AF which may be derived from a UV-light induced tryptophan photodegradation reaction or by non-enzymatic glycosylation of the lens proteins.
We have developed an objective, non-invasive and highly reproducible technique to
measure blue-green AF of the lens. The lens AF profile can also be used for evaluating
other lens transmission properties, such as scattering and absorption of light. In this study
the changes in lens AF with ageing and cataractogenesis were examined. The results were
compared with the widely used subjective lens opacities classification system (LOCS II
and III) and with the back light scatter values obtained by the commercially available
Lens Opacity Meter (LOM). The limitations of the method and the means to improve our
fluorometer are discussed.
The values of blue-green AF and light scatter (LOM) in the lens increased in a very
similar fashion with age in healthy eyes. According to the regression lines AF begins to
increase in early childhood. It appears by extrapolation to be absent at birth. In contrast
light scatter in the lens was present even in young children. Elderly subjects in the 8th
decade showed almost a 4 times greater maximum AF in the lens compared to individuals
in the 3rd decade of life. The lens transmission index, quantified from the lens AF curve,
was almost unchanged up to 60 years of age. Thereafter it decreased rapidly and the interindividual variation of lens transmission properties increased.

70
In cataractous lenses the mean AF and scatter values differed statistically significantly
from those of age matched healthy controls. The highest AF values were measured in
nuclear cataracts where AF was also found to be related to visual acuity and an increasing
yellow-brown colour of the nucleus. About half of the total variation of the transmission
index values could be accounted for by changes in nuclear colour as assessed by the
LOCS III grading system. In cortical cataract the AF curve was low and flattened and the
maximum AF value was significantly lower than in the control eyes. On the other hand
the highest light scatter values were measured in the cortical cataracts. However, the correlation between LOCS III cortical grades and light scatter values was rather weak.
We could demonstrate how lens yellowing determined with the lens AF had an actual
effect on RNFL visibility in black-and-white images taken with a blue monochromatic filter. The regression analysis indicated that the lens AF measurements provided a better
prediction about the RNFL visibility score than age or visual acuity did.
Many investigators have proposed an UV-light induced photo-oxidative mechanism
for cataract formation. Partly the low glutathione concentration in the lens nucleus makes
it particularly vulnerable to long-term photo-oxidative stress following crystallin aggregation, pigmentation and development of non-tryptophan fluorescence. These theories and
the increase in solar UV exposure due to the thinning of the ozone layer gives us a good
reason to continue our studies. Measurements of corneal and lens AF will be of particular
interest, because of their clear association with morphological and biochemical changes
in these tissues.
In conclusion, the results of the present study suggest that the lens fluorometry may be
a useful additional tool together with a subjective grading system in the follow-up of optical changes occurring in the nuclear region of the lens. It may also be a practical way of
determining the amount of yellow-brown coloration of the lens.

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