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Circadian field photometry December 1, 2006
PETTERI TEIKARI [email protected]
PROJECT WORK OF MEASUREMENT SCIENCE AND TECHNOLOGY FOR THE
COURSE S-108.3120 PROJECT WORK
Course credits:
ECTS Points
Grade (1-5):
Supervisors signature:
M.Sc. Tuomas Hieta
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Symbols and
abbreviations.................................................................................................
3 1. Introduction
............................................................................................................
4 2. Circadian photobiology
.........................................................................................
6
2.1 Circadian rhythms
.........................................................................................................7
2.2 Circadian clock mechanism
..........................................................................................8
2.3 Physiology of the eye
....................................................................................................9
2.3.1 Ophthalmological optics
.....................................................................................................
12 2.3.2 Pupil pathways
....................................................................................................................
15 2.3.3 Eye movements
...................................................................................................................
17
2.4 Light characteristics
....................................................................................................19
2.4.1 Spectrum
.............................................................................................................................
19 2.4.2 Spatial distribution
..............................................................................................................
22 2.4.3
Intensity...............................................................................................................................
23 2.4.4
Timing.................................................................................................................................
24 2.4.5 Duration
..............................................................................................................................
25 2.4.6 Photic history
......................................................................................................................
26 2.4.7 Polarization
.........................................................................................................................
26
3. Eye and photometric measurements
..................................................................
27 3.1 Electrical activity
........................................................................................................27
3.1.1 Electroretinogram (ERG)
....................................................................................................
27 3.1.2 Electooculogram
(EOG)......................................................................................................
29
3.2 Eye tracking
................................................................................................................30
3.2.1 Pelz et al. (2000, 2004)
.......................................................................................................
32 3.2.2 Li et al. (2006): openEyes
...................................................................................................
33
3.3 Pupil size
.....................................................................................................................37
3.3.1 Video-driven infrared pupillography
..................................................................................
38 3.3.2
Photorefractometry..............................................................................................................
40 3.3.3 Digital photography
............................................................................................................
41
3.4 Digital-imaging circadian photometry
........................................................................43
3.4.1 Circadian-weighed luminancephotometers (Gall et al., 2004)
............................................ 43 3.4.2 Digital
photography (Hollan et al., 2004)
...........................................................................
45
3.5 Dosimeters
..................................................................................................................47
3.5.1 LichtBlick (Hubalek et al.,
2004)........................................................................................
48 3.5.2 Daysimeter (Bierman et al., 2005)
......................................................................................
49
4. Dosimeter design and
simulation........................................................................
54 4.1 Eyetracker and/or pupil size
measurement..................................................................54
4.2 Dosimeter
....................................................................................................................56
4.2.1 Photodiode-based dosimeter
...............................................................................................
56 4.2.2
Spectroradiometer-based.....................................................................................................
61
5. Conclusions
...........................................................................................................
63 6. References
.............................................................................................................
65
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Symbols and abbreviations 3
SYMBOLS AND ABBREVIATIONS wavelength max peak wavelength W/cm2
microwatt per square centimeter A/Hz amperes per root hertz A/W
amperes per watt acv circadian action factor Ap pupil area Ar area
of the image at the retina As source area B noise bandwidth [Hz]
B() action spectra for blue light hazard (ICNIRP) B()
biological/circadian action spectra b-lx blue-lx, unit for
blue-colored illuminance BY hypothetical luminous efficiency
function for
circadian responses. c() circadian action function cd/m2
candelas per square meter, unit for luminance dB decibel DC direct
current dp pupil diameter dr diameter of the image at the retina ds
diameter of the source E Energy Ec corneal irradiance en noise
voltage density Er retinal irradiance f frequency f focal length g
gram h Plancks constant Hg high-pressure mercury Hz Hertz, unit for
frequency Id dark current If feedback (gain) current Ijn Johnson
noise current In noise current in noise current density In,e noise
current from en in,e noise current density from en Ip photocurrent
Itot total noise current J/cm2 Joules per square centimeter K
Kelvin, unit for (color) temperature kB Boltzmann constant kbauds/s
kilobauds per second lm/w lumens per watt, unit for luminous
efficacy Ls source radiance lx lx, unit for illuminance mA
milliampere mAh milliampere hour MB megabyte Mbps megabits per
second MHz megahertz mm millimeter M melanopsin-containing retinal
ganglion cell
spectral efficiency nm nanometer, normal unit for wavelength nV
nanovolt pA picoampere Pr retinal power P spectral irradiance at
the eye [W/m2/nm] Rf feedback (gain) resistance Rsh shunt
resistance R photosensitivity S S cone spectral efficiency T
temperature [K] V()
spectral sensitivity curve for photopic vision V/Hz volts per
root hertz V rod spectral efficiency function V10 photopic spectral
sensitivity for centrally fixated
large target
VDC volts, direct current W watt Xec circadian radiation
quantity Xv photometric radiation quantity angular subtense of the
source lens transmittance luminous flx [lm] e, Spectral radiance
[Wm-2] ohm, unit for resistance s solid angle [sr]
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Symbols and abbreviations 4
+/+ wild type mice ADC analog-to-digital converter AgCl silver
chloride CBT core body temperature CBTmin core body temperature
minimum CCD charge-coupled device CCT correlated color temperature
CD compact disc CIE International Commission on Illumination CMOS
complementary metal oxide semiconductor CP constant posture CR
constant routine CRH corticotropin-releasing hormone CT circadian
time DLMO dim light melatonin onset DLMOff dim ligh melatonin
offset DLMOn dim light melatonin onset DMH dorsomedial hypothalamic
nucleus dmSCN dorsomedial suprachiasmatic nucleus dSPZ dorsal
subparaventricular zone EB eye blink ECG electrocardiogram EEG
electroencephalogram EOG electrooculogram ERG electroretinogram ERP
early-receptor potential EW Edinger-Westphal nucleus fMRI
functional magnetic resonance imaging FOV field of view FWHM full
width at half maximum GNU GNU's Not Unix GPL General Public License
GTP ganosine triphosphate hbw half bandwidth hbw half bandwidth IC
integrated circuit IEEE-1394 Institute of Electrical and
Electronics Engineers,
standard 1394 (FireWire, i.LINK) INL inner nuclear layer ipRGC
intrinsically photosensitive retinal ganglion cell IR infrared IRC
intensity response curve IRED infrared LED KRG potassiumretinogram
LASIK laser-assisted in situ keratomileusis LCD liquid crystal
display LED light emitting diode LGN lateral geniculate nucleus LRP
late-receptor potential MPO medial preoptic region mRGC
melanopsin-containing retinal ganglion cells M-RGC magno-retinal
ganglion cells NIF non-image forming ONL outer nuclear level PF
prefrontal PLR pupillary light reflex PPRF paramedine pontine
reticular formation PRC phase response curve P-RGC parvo-retinal
ganglion cells PRK photorefractive keratectomy PU Pupillary Unrest
PUI Pupillary Unrest Index RANSAC Random Sample Consensus REM rapid
eye movement sleep RGC retinal ganglion cell RHT
retino-hypothalamic tract RI retinal illuminace RMS
root-mean-square RS-232 a standard for serial binary data
interconnection SCN suprachiasmatic nucleus SD standard deviation
SEM slow eye movement SEM standard error of the mean SPD spectral
power distribution USB Universial Serial Bus
UV ultraviolet VDT video display terminals VDU video display
unit VLPO ventrolateral preoptic nucleus vlSCN ventrolateral
suprachiasmatic nucleus vSPZ ventral subparaventricular zone
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Introduction 5
1. INTRODUCTION
Over 150 years since the discovery of the retinal rod and cone
photoreceptors in 1834, it has been believed that, both visual and
biological effects induced by light would be dependent on those two
traditional photoreceptors. However in 2002, through the discovery
of a novel photoreceptor in the eye by David Berson et al. [1]
views have changed on how human vision system works. The novel
photoreceptor, intrinsically photosensitive retinal ganglion cell
(ipRGC) is one of the known ~20 ganglion cells in human retina. It
has been estimated that of all retinal ganglion cells (RGC), 0.25%
are photosensitive ipRGCs [2]. It was found [3] that the novel
photoreceptor is responsible mainly for regulating light-induced
human biological rhythms (circadian rhythms) by synchronizing body
to environmental light/dark-cycle. It has also been proposed [4] to
mediate light-induced increase in alertness, pupillary responses,
and as a possible target for seasonal depression treatment. This
novel photoreceptor may have many consequences for practical
applications both in general lighting and lighting for special
groups (e.g. elderly, shift workers, patients suffering from
seasonal depression) so it has become a great interest of research
in the lighting community [5]. However, the characteristics of
non-image forming (NIF) visual system differ from the conventional
visual system based on cones and rods. The NIF visual system has a
higher threshold for activation, requires longer exposures for
activation, depends on the location of light source in the visual
field, and most importantly has different spectral characteristics
with the peak wavelength (max=480 nm) shifted towards the blue part
of visible spectrum. Given that the spectral characteristics are
different with the NIF visual system; conventional photometric
illuminance can not be used to quantify the NIF responses in
humans. As the temporal characteristics (duration and timing of
light exposure) differ from visual system, simple measurement of
task illuminance is not sufficient to determine the NIF effective
light response for example during normal office work. Measurement
situations with NIF effective lighting can be divided roughly into
two categories: measurement in strictly controlled laboratory
studies and to field studies and workplace measurements. This work
addresses the problems measuring the NIF effective light exposure
by means of a literature review in field conditions using a
portable head-mounted light dosimeter. However given that the
biological effects of given light exposure are not completely
unknown, the simple measurement of light exposure is not sufficient
to produce new knowledge on the NIF visual system. Both in field
and laboratory studies physiological measurements are needed to
study the causality between biological effects and light. Within
the scope of this work the physiological measures are only briefly
reviewed. In reality when designing a dosimeter, the simultaneous
measurement of measures such as ERG, EOG, EEG and ECG with light
exposure should be taken into account (e.g. electromagnetic
compatibility and space/weight restrictions). For a review of the
physiology related to light exposure and NIF responses, the Masters
thesis of the author is suggested [6]. In this work chapter 2
reviews briefly the circadian photobiology that is needed to
understand the needs of the new measurement device needed to
quantify the NIF responses. Chapter 3 reviews the literature on
existing technologies for measuring NIF effective light exposure
and the related measurements such as pupil size, eye tracking,
electrical activity of eye, and NIF effective ambient luminosity
distribution. In Chapter 4 the possible improvements and
cost-cutting methods for the measurement are reviewed with some
basic signal-to-noise ratio comparisons.
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Circadian photobiology 6
2. CIRCADIAN PHOTOBIOLOGY
The whole concept of circadian rhythm is the most essential
point of this work so its essential to clarify what it really
means. Circadian (circa, meaning approximately and dies, meaning
day) clock exists practically in every organism starting from
cyanobacteria to humans [7-10]. Genuine circadian rhythms are
generated totally endogenously without external cues
(zeitgebers=time givers) like light. Light is thought to be the
strongest zeitgeber, but in reality some weaker zeitgebers
(nonphotic entrainment) can have even greater effect on human
circadian rhythms in certain special cases. These weaker zeitgebers
include social interaction, sleep/wake schedules, food, drugs,
auditory and olfactory stimuli, temperature, and exercise [11]. In
general circadian rhythms are advantageous to organisms to
anticipate changes in the environment, such as the rising and
setting of the sun. Other rhythms that affect our bodies include
ultradian, which are cycles that are shorter than day, for example,
the milliseconds it takes for a neuron to fire, or a 90-minute
sleep cycle. Theres also infradian, referring to cycles longer than
24 hours like monthly menstruation for example. There are also
seasonal rhythms (photoperiod) like those in hibernation, and
reproduction [12]. People have noted the existence of daily rhythms
throughout the history. In 1729, Jean Jacques DOrtous de Mairan, a
French astronomer, had a sharp insight into how to test whether a
daily rhythm is internal or completely dictated by external
stimulus [13]. He studied this by keeping a heliotrope (plant) in
the dark and noticed that its leaves kept opening and closing
despite the lack of light. De Mairan however didnt conduct any
further experiments on this subject. In the 1850s Karl von Frisch,
Gustav Kramer and Colin Pittendrigh each independently discovered
compelling evidence for internal clocks in animals [14]. These
investigations were the beginning of the modern field of circadian
photobiology. In the 1960s Jrgen Aschoff, a German researcher
conducted a human study on circadian rhythms [15]. He built a
bunker in which volunteers lived for several weeks. Most of the
time they were totally isolated from real world without any
sunlight, and occasionally the door to bunker was opened revealing
the real world. Aschoff noticed in his study that even though
volunteers were isolated, their sleep-wake cycles persisted. But he
also found that their biological clock got desynchronized with the
real world because their endogenous cycle wasnt exactly 24 hours,
average periodicity being 24.5 hours. Aschoffs final conclusion was
that when the door was reopened, the subjects readjusted their
clocks meaning that it was possible to reset their circadian clock
by external cues. Still Aschoff didnt know how this circadian clock
works, and whether it could be located in a particular part of the
body. Normally animals have been divided into nocturnal (active
during night) and diurnal (active during day), but there exists
also some adaptability to environmental situation at circadian
level, which is the case with Finnish bats which are nocturnal
mainly during the warm months of the summer. However, in the spring
and fall, when environmental factors no longer favor night flight
(fewer insects to eat during the colder nights, and fewer birds to
compete with and to prey on bats during the day), their activity
cycle shifts to the daylight hours [16,17]. In the 1970s scientists
demonstrated that the mammalian clock is located in a part of the
brain called hypothalamus, specifically in a set of neurons on each
side of the
Figure 1. Rhythm of Finnish bats. The gray region indicates the
night period and the black bars show the actual activity time for
the bats on each day [17].
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Circadian photobiology 7
brain called the suprachiasmatic nucleus (SCN). It was noticed
that when removing the SCN from rats and hamsters, the animals lost
their normal biological rhythms. However when transplanting the SCN
back to into hamsters, the normal rhythms were restored. Recently
experiments have shown that the SCN can be completely isolated from
the animal and still measure chemical and physiological signals
from it in vitro conditions. Later SCN and the other hypothalamic
nuclei involved in regulation of circadian rhythms will be
reviewed.
2.1 CIRCADIAN RHYTHMS Cyclical fluctuations around 37C in core
body temperature (CBT) are perhaps the best documented circadian
rhythm. Gierse [18] had already shown in 1842 that his own oral
temperature revealed a maximum temperature in the early evening and
minimum in the early morning. Aschoff et al. [19,20] showed that
circadian rhythm is caused both by changes in heat production and
changes in heat loss, and concluded that heat production undergoes
a circadian rhythm which is phase advanced by 1.2 h with respect to
the circadian rhythm of heat loss, and this delay is caused by
bodys inertia and because transport of heat takes time. This
individual regulation of the heat production and heat loss results
in much finer tuning of the CBT rhythm than if only one of these
components were regulated [21]. It has been proposed [22] that body
temperature represents the underlying mechanism regulating
performance. The speed of thinking and performance depends on the
level of metabolic processes in neurons in the cerebral cortex.
However, the interrelationship between thermoregulatory and
sleepiness/performance regulatory mechanisms is rather complex and
not fully understood [21]. CBT can be easily measured continuously
by using a rectal thermistor (e.g. Harvard Apparatus YSI 400 Series
[23]). Another common circadian rhythm measured in chronobiological
studies, is the circadian rhythm of melatonin hormone. Melatonin
(C13H16N2O2; molecule weight232,278 g/mol),
5-methoxy-N-acetyltryptamine, is a hormone produced primarily by
pinealocytes in the pineal gland (located in the brain) [24]. It
can be considered to be a reliable marker of the circadian phase as
it is secreted in very strict circadian manner peaking during the
night. It is synthesized and secreted at night in both day-active
and night-active species [25], thereby acting as a signal for the
length of day and night. Despite its robust circadian behavior many
mechanisms of melatonin are still unclear [26]. Abnormal melatonin
levels caused by lighting at wrong biological time in night-shift
workers have been connected to increased risk of breast cancer in
women [27-30]. The typical circadian variations of plasma melatonin
and core body temperature are seen in Figure 2 [31]. Melatonin
levels are usually used as a
Figure 3. Relative (%) circadian phase markers using melatonin.
DLMO, dim-light melatonin onset, DLMOFF dim-light melatonin offset
[33].
Figure 2. Plots of (A) endogenous plasma melatonin, and (B) core
body temperature with data folded at endogenous circadian period as
determined by core body temperature for each subject. Abscissa
refers to biological time which corresponds different clock time in
every individual. In average minimum in CBT (CBTmin) occurs around
04:00 hours [31].
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Circadian photobiology 8
marker for the phase shifts in circadian rhythms. This
phase-shift in practice means that light exposure can delay or
advance the onset of nocturnal melatonin rhythm [32]. Typical
methods to assess the time of nocturnal melatonin surge can be seen
in Figure 3 [33]. Other circadian rhythms include the diurnal
rhythms of cortisol [34], thyrotropin (TSH) [35], prolactin [36],
vasopressin [37], and growth hormone (GH) [38] among many
others.
2.2 CIRCADIAN CLOCK MECHANISM In this chapter different brain
regions and the hormones involved in the regulation of circadian
rhythms are briefly reviewed. The information presented here should
be taken with caution as all the presented areas require further
research. Hypothalamus is a structure in the brain located below
the thalamus and it regulates various metabolic and autonomic
processed [39]. Given its central position in the brain and its
proximity to the pituitary (Figure 4) it is involved as an
integrator of both sensory and contextual information. Hypothalamus
consists of various nuclei (Figure 5). A lot about the hypothalamus
is still unknown, but some actions are at least partially
understood and can be described at basic level. Suprachiasmatic
nuclei (plural form of nucleus) are nuclei in the hypothalamus
situated immediately above the optic chiasm (Figure 5) on either
side of the third ventricle in anterior hypothalamus. The SCN is
one of
four nuclei that receive nerve signals directly from the retina
through retinohypothalamic tract (RHT, Figure 4); the others are
lateral geniculate nucleus (LGN), the superior colliculus and the
pretectum. In the 1970s the biological clock was located in SCN
[40,41], and it was shown that SCN contain genetically driven clock
mechanism that ensures a nearly 24 hour cycle [42]. Precise
estimation of the periods of the endogenous circadian rhythms of
melatonin, core body temperature, and cortisol in healthy
individuals living in carefully controlled lighting conditions
indicates that the intrinsic period of the human circadian
pacemaker averages 24.18 hours with a tight distribution that is
consistent with other species [43]. Circadian rhythmicity is
abolished by SCN lesions [41] and restored by SCN transplants [44].
Traditionally SCN has been subdivided into a dorsomedial shell
(dmSCN) and a ventrolateral core (vlSCN) based on retinal
innervation and phenotypically distinct cell
Figure 5. The hypothalamus, showing the location of the
suprachiasmatic nucleus (SCN), which in mammals is the primary
biological clock. [39]
Figure 4. Schematic summary of targets influenced by
photosensitive retinal ganglion cells. Projections to the SCN from
the retinohypothalamic tract (RHT) [39].
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Circadian photobiology 9
types [45,46], while this subdivision has also been criticized
for simplifying the SCN structure [47]. Intrinsically rhythmic
cells are largely confined to the SCN shell [48], receive little
retinal innervation [46], and displays delayed clock gene
expression following phase-shifting light exposure [49]. Cells in
the SCN core receive direct retinal innervation [50] and express
c-fos, Per1 and Per2 in response to phase-shifting light pulses
[51-53]. Cells in the SCN core oscillate in response to light
stimulus. Light exposure always increases firing rates in SCN
neurons [54], although light induces clock gene expression in the
SCN only during the night [55]. The simplified assumption that SCN
is responsible solely for circadian rhythms is inadequate for
in-depth understanding of the human circadian rhythms. Currently
human circadian rhythms are thought to be controlled via
multioscillator organization hypothalamic nuclei [56-58]. SCN
provides three major output pathways. One pathway runs into the
medial preoptic region (MPO) and then up into paraventricular
nucleus of thalamus. A second pathway runs to the retrochiasmatic
area and the capsule of the ventromedial nucleus. The third
pathway, which contains the largest portion of the SCN efferent
(going away, opposite is afferent) flow, runs mainly to vSPZ and
dSPZ with smaller proportion terminating to the DMH. Also small
numbers of SCN axons innervate directly the areas that are involved
in feeding, wake-sleep cycles and secretion of hormones such as
melatonin (presumably through dorsal parvicellular portion of the
paraventricular nucleus [59]) and corticotrophin-releasing hormone
(CRH) [60]. The further examination of circadian clock mechanism is
beyond the scope of this work.
2.3 PHYSIOLOGY OF THE EYE The simplified anatomy of an eye is
shown n Figure 6 [61]. The pupil allows light to enter the eye. It
appears dark because of the absorbing pigments in the retina. The
pupil is surrounded by beautifully pigmented iris, which is a
circular muscle controlling the amount of light entering the eye.
Both pupil and the iris are covered by a transparent external
surface called the cornea. This is the first and most powerful lens
of the optical system of the eye and allows, together with the
crystalline lens the production of a sharp image at the retinal
photoreceptor level. The purpose of the lens is to focus light onto
the back of the eye. The lens is encased in a capsular-like bag and
suspended within the eye by tiny guy
wires called zonules. The cornea is continuous with the sclera,
the white of the eye, which forms part of the supporting wall of
the eyeball. Furthermore this external covering of the eye is in
continuity with the dura of the central nervous system. The sclera
and the cornea form the external layer of eye.
Figure 6. a) Vertical, and b) horizontal sagittal section of the
adult human eye [61].
a) b)
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Circadian photobiology 10
Retina is the sensory part of eye and part of the central
nervous system. The central point for image focus (the visual axis)
in the human retina is the fovea. The optic axis is the longest
sagittal distance between the front or vertex of the cornea and the
furthest posterior part of the eyeball. It is about the optic axis
that the eye is rotated by the eye muscles. In the center of the
retina is the optic nerve, a circular oval white area. From the
center of the optic nerve radiate the major blood vessels of the
retina. Approximately 17 degrees (4.5-5 mm), or two and half optic
disc diameters to the left of the optic disc (or optic nerve head
is the point in the eye where the optic nerve fibers leave the
retina), can be seen the slightly oval-shaped, blood vessel-free
reddish spot, the fovea, which is at the center of the area known
as the macula. It is a small and highly sensitive part of the
retina responsible for detailed central vision. A circular field of
approximately 6 mm around the fovea is considered the central
retina while beyond this is peripheral retina stretching to the ora
serrata. The optic nerve contains the ganglion cell axons running
to the brain and, additionally, incoming blood vessels that open
into the retina to vascularize the retinal layers and neurons. A
radial section of a portion of the retina reveals that the ganglion
cells (the output neurons of the retina) lie innermost in the
retina closest to the lens and front of the eye, and the classical
photosensors (the rods and cones) lie outermost in the retina
against the pigment epithelium and choroid (Figure 7A [62]). All
vertebrate retinas are composed of three layers of nerve cell
bodies and two layers of synapses (Figure 7B). The outer nuclear
layer (ONL) contains cell bodies of the rods and cones, the inner
nuclear layer (INL) contains cell bodies of the bipolar, horizontal
and amacrine cells and the ganglion cell layer contains cell bodies
of ganglion cells and displaced amacrine cells. Between these
layers are areas called neuropils where synaptic contacts
occur.
Traditionally cones and rods have been thought to be the only
photoreceptors in mammalian retina, but after the discovery of the
novel photoreceptor ipRGCs the exact roles of all three
photoreceptors are not fully understood. The rod system is
specialized for vision at very low light levels, but with the
expense of poor spatial resolution. When only rods are activated
the perception is called scotopic vision. With only rods active it
is impossible to neither sense color differences or to make exact
visual discriminations. The cone system has a very high spatial
resolution, with color sensing abilities in the expense of poor
light sensitivity. At about the level of starlight the cones begin
to contribute to vision and they become more and more dominant as
light level increases. At very high light levels such as in
sunlight, only cones are active and rods are totally saturated
[63]. This condition is called photopic vision. The area between
scotopic and photopic vision is called mesopic
(A)
Figure 7. (A) Simple diagram of the organization of the retina.
(B) 3-D block of a portion of human retina. [62]
(B)
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Circadian photobiology 11
vision, which is characterized by contribution of both rods and
cones. The estimated upper luminance limit for mesopic vision is
3-10 cd/m2 [64]. Spectral sensitivities for photopic, mesopic and
scotopic vision can be seen in Figure 8 [65]. The retina contains
about 20 different retinal ganglion cells (RGCs) [66], which
basically are responsible for the output of visual data to the
brain. At the basic level ganglion cells can be divided in two
ways, either by their receptive field with the division to magno
(M-) and parvo (P-) cells, or by their polarization response to
light (ON and OFF cells). M-RGCs terminate in the magnocellular
layer of the lateral geniculate nucleus (LGN) of the thalamus, and
P-RGCs terminate in the parvocellular layer of the LGN. The
conventional view was that ganglion cells got their commands from
rods and cones, and ganglion cells did not have any light-sensitive
properties themselves [1]. In the beginning of 1980s, however,
behavioral studies especially those of Foster and colleagues, began
to challenge this model [67]. Photic entrainment exhibited high
thresholds, low-pass temporal filtering and long-term temporal
integration that seemed difficult to explain with the conventional
model of cones and rods. This was backed up by studies made with
blind mice [68-70] with severe degeneration of classical
photoreceptors as well as studies done with certain blind humans
[71]. However, it was not clear at all that the receptor for
circadian phase would be found from the eye. In non-mammalian
animals light penetrating directly to brains acts as circadian
pacemaker. In mammals, however, many studies were made and no
impact on circadian phase could be shown after eye removal [72-75].
Interestingly, one study reported a bright light behind the knee
phase-shifting circadian rhythm [76], but the results could not be
replicated [77,78] making this explanation a bit unlikely at the
moment. The discovery of circadian photoreceptor was at last made
by David M. Berson et al. [1]. The novel photoreceptor is
abbreviated as ipRGC (intrinsically photosensitive retinal ganglion
cells), or as mRGC (melanopsin-containing retinal ganglion cell,
mRGC) due to the photopigment responsible for the noticed non-image
forming (NIF) effects. Melanopsin was first discovered by Ignacio
Provencio and his colleagues [79,80], and is named by the cells in
which it first was isolated: the dermal melanophores of frog skin.
The two main differences of ipRGCs compared to cones and rods, are
that light depolarizes ipRGC while the opposite happens with rods
and cones; and ipRGCs are far more sluggish compared to rod and
cones, response latencies being as long as a minute. The results
are not consistent about the peak wavelength of melanopsin-pigment.
Qiu et al. [81], and Panda et al. [82] show that melanopsin max is
very close to 480 nm, but Melyan et al. [83] and Newman et al. [84]
suggest that melanopsin has max closer to 420-430nm. The most
likely explanation for this kind of large difference was that
Newman et al. [84] were the only ones who determined the direct
absorption spectrum of melanopsin in vitro conditions whereas all
the other studies were done in vivo conditions [85]. Peak
absorption spectrum of 420-430 nm might well be the intrinsic peak
wavelength for melanopsin, but it would not be the actual peak
wavelength responsible of the wide range of the biological effects
mediated by ipRGCs. There is also some preliminary evidence that
some cones contain also melanopsin and are involved in circadian
phototransduction [86].
Figure 8. Spectral sensitivity functions of the eye. In photopic
vision, when cones are active, the sensitivity follows the function
V() with a peak wavelength of 555nm. At very low light levels only
rods are active, and spectral sensitivity follows V()-function with
a peak wavelength of 505nm. The Vmes() is one example of the
possible mesopic spectral sensitivity as no consensus exists on it
yet. The V10() is is the photopic spectral sensitivity for
centrally fixated large target [65].
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Circadian photobiology 12
2.3.1 Ophthalmological optics
Figure 9 shows the human visual fields, which are divided first
to monocular (one eye) and binocular visual fields (two eyes), and
then further into superior/inferior and nasal/temporal visual
fields. Figure 9B shows how the image is inversed onto the surface
of the retina, and how the different quadrants of monocular visual
fields are related to the binocular vision. It is important to
notice from Figure 9C that light reaching nasal (inner) part of the
retina is coming from peripheral visual field and vice versa, and
the same thing happens with superior/inferior visual fields, where
the object (e.g. sky) in the superior visual field is projected to
the inferior part of the retina [39]. Binocular visual field is
larger than either of the monocular visual fields. Forehead, nose
and cheeks limit visual field so that it is larger horizontally
than vertically. Binocular visual field is horizontally about 190,
and below the horizontal level about 70-80 and above 50-60 [87]. It
should be noted that the human visual field is much larger than
normal 35mm lens used in cameras. However, visual processing is not
uniform across the visual field: 25% of cortex is devoted to the
central five degrees of the field of view [88].
Figure 9. Projection of the visual fields onto the left and
right retinas. (A) Projection of an image onto the surface of the
retina. The passage of light rays through the optical elements of
the eye results in images that are inverted and left-right reversed
on the retinal surface. (B) Retinal quadrants and their relation to
the organization of monocular and binocular visual fields, as
viewed from the back surface of the eyes. (C) Projection of the
binocular field of view onto the two retinas and its relation to
the crossing of fibers in the optic chiasm. Points in the binocular
portion of the left visual field (B) fall on the nasal retina of
the left eye and the temporal retina of the right eye. Points in
the binocular portion of the right visual field (C) fall on the
nasal retina of the right eye and the temporal retina of the left
eye. Points that lie in the monocular portions of the left and
right visual fields (A and D) fall on the left and right nasal
retinas, respectively. [39].
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Circadian photobiology 13
Human eye as an optical instrument is briefly reviewed here as
the actual retinal illuminance or irradiance depends on the optical
characteristics of the eye. Figure 10B [89] shows the simplified
version of the human eye as an optical system. The size of the
pupil determines (pupil diameter dp) the light entering the eye.
Figure 10A [89] shows the wavelength dependent transmittance (from
cornea to retina) and retinal absorption. Transmittance is
important for the actual retinal irradiance whereas retinal
absorption affects the amount of retinal damage from light
exposure.
Between the corneal irradiance Ec, the retinal irradiance Er,
and the radiance of the source Ls, the following relation exists
[89]:
p
rr
r
r
2s
2
2p
2s
2p
2r
2s
sssc AAE
AP
r
Afd4
r
Ad
f4Er
ALLE
=
pi=
pi=== (1)
r
pcs2
pr A
AEL
f4d
E
=
pi= (2)
Where, Ec = corneal irradiance Ls = source radiance s = solid
angle [sr] As = source area r = distance between the source and
lens Pr = retinal power = lens transmittance dp = pupil diameter f
= focal length, can be estimated to be 1,7 cm [89,90] Er = retinal
irradiance Ar = area of the image at the retina Ap = pupil area
The image size (diameter) of the source at the retina can be
calculated quite simply [89]:
fr
fdd sr == (3)
Where, dr = size of image at the retina ds = size of the source
f = focal length, can be estimated to be 1,7 cm [89,90] r =
distance between the source and lens = angular subtense of the
source
Transmittance
Absorption
%
Figure 10. (A) Transmittance of optical radiation from cornea to
retina and the absorption at the retina [89]. (B) Eye as an optical
system [89].
(A) (B)
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Circadian photobiology 14
The size of the image at the retina depends on distance between
the source and lens. In practice the retinal irradiance can de
different while the corneal irradiance is the same. Larger angular
subtense produces smaller retinal irradiance and smaller then
consequently produces larger retinal irradiance [91]. This is why
the corneal irradiance (or illuminance) should always be controlled
when conducting experiments by neutral density filter rather than
moving light sources further from eye as circadian responses
ultimately depend on the retinal irradiance. Figure 12 shows the
wavelength dependent average transmittance of human lens [92-95].
It can be seen that in the visible part of spectrum newborn lens
does not have significant wavelength dependence. In the age group
of 20-29 years the transmittance of the blue part of the visible
spectrum is slightly attenuated, and in the age group 60-69 years
the attenuation is really significant due to yellowing of the lens.
In visual responses the human brain can compensate the attenuation
of the blue light in a manner that the world does not appear to be
less blue for the older people [96,97]. The transmittance of the
human lens in different age groups accompanied with the spectral
transmittance of intraocular lens (used after cataract removal
surgery) [98], the proposed melatonin suppression curve, and the
cornea is shown in Figure 11 [99]. Corneal spectral transmittance
remains relatively constant in aging as supported by the study by
Beems et al. [100] that the corneal transmission for donors younger
than 45 yr (n = 3, 2243 yr) did not differ significantly from that
of donors older than 45 yr (n = 5, 6787 yr) at any wavelength.
Figure 12. The average transmittance of human lenses for three
different age groups as a function of wavelength [92,95].
WAVELENGTH
%
Figure 11. Transmittance data for lens: 14 years (); 49 years
(+); 92 years () (after Weale, 1985 [93]); mean lens data (X)
(after Stockman and Sharpe, 2000 [63]); intraocular lens (*) (after
Mainster, 1986 [98]); cornea () (after Beems and van Best, 1990
[100]). The heavy continuous curve shows the relative sensitivity
of the presumed photopigment (after Thapan et al., 2001 [116].
Graph from Charman [99].
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Circadian photobiology 15
2.3.2 Pupil pathways
It was assumed in earlier days that pupil light response (PLR)
was driven by a single subcortical pathway and this was because of
persisted pupillary light reflex in cortically blind people [101].
Recently however this hypothesis has been replaced with a theory of
bilateral signaling involving two different pathways [102].
Pupillary reflexes have been divided into steady-state pupil size
depending on the ambient light level, and brisk and transient
constriction of pupil size depending on rapid changes in light
flux, which is also described as dynamic PLR response. Shining
light in the eye thus leads to an increase in the activity of
pretectal neurons, which stimulates the Edinger-Westphal neurons
and the ciliary ganglion neurons they innervate, thus constricting
the pupil. Pupil-related pathway is shown in Figure 13.
Pupil size is determined by iris movement, which is controlled
by two antagonistic muscles, the sphincter and the dilator.
Activation of the sphincter of the iris causes the pupil to
constrict (i.e., miosis), this being largely under parasympathetic
control and involving ciliary ganglion. Dilator is under
sympathetic control, and causes the pupil to dilate (mydriasis)
through superior cervical ganglion controlled by EW nucleus.
Sympathetic system is associated with fight-or-flight responses
with epinephrine and norepinephrine stimulation. Parasympathetic
system is the opposite and it is sometimes called the rest and
digest system for its ability to relax and slow down the functions
of organs (slowing heart beat and increasing its constricting
power). In practice general arousal through increased sympathetic
activity will cause pupil dilation independent of the ambient light
level, and vice versa. Like with all lens systems, the size of the
pupil
Figure 13. The circuitry responsible for the pupillary light
reflex. This pathway includes bilateral projections from the retina
to the pretectum and projections from the pretectum to the
Edinger-Westphal nucleus. Neurons in the Edinger-Westphal (EW)
nucleus terminate in the ciliary ganglion, and neurons in the
ciliary ganglion innervate the pupillary constrictor muscles.
Notice that the afferent axons activate both Edinger-Westphal
nuclei via the neurons in the pretectum [39].
Figure 14. Example of dynamic pupil light reflex responses to
flashes of increasing luminance contrast, i.e., L/Lb = 0.3, 0.6,
0.9, 1.2, 1.5 & 2.15 [102].
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Circadian photobiology 16
(aperture) determines the amount of light entering to retina
(retinal illuminance, RI). Pupil size also controls the aberrations
and depth of the field of the eye in the same manner as in camera.
A smaller aperture (larger f-value) will enhance depth of field and
reduce aberrations [103]. PLR response can be divided into
steady-state and transient (dynamic) component. The steady-state
component is determined by ambient light level and is characterized
by neural mechanisms that response to overall light flux changes,
large dynamic range and exhibit large spatial summation. Dynamic
PLR response is very rapid to rapid light flux change as seen in
Figure 14 [102]. Observed transient constriction would need from
neurons the following properties: limited spatial summation,
band-pass temporal response characteristic, and high contrast gain.
A light stimulus always depends on both components, but the
relative contribution each component makes to constriction will
depend on size of the stimulus, its luminance contrast, onset
temporal characteristics and location in the visual field. As
observed in Figure 14 pupil constriction is greater with higher
luminance contrast, when pupil response is more dominated by
steady-state component. Participation of steady-state component
could be even further increased with larger stimulus size. Despite
involvement of both rod and cone photoreceptors in determining
pupil size [104-106], there is an increasing amount of evidence
pointing out that ipRGCs play some role in pupillary controls as
functional pupillary light reflex (PLR) has been shown to be
retained in rodent models of retinal generation (impaired cone/rod
function) [107-111]. The results of the spectral sensitivity for
the pupillary reflex obtained by Alpern and Campbell [105] can be
seen in Figure 15. It can be seen that photopic pupil response is
close to the photopic spectral efficiency curve V() and the
scotopic pupil response curve is close to the scotopic spectral
efficiency curve V(). However, pupil size has been noticed to be
smaller under light with higher CCT [136] (8000 K) compared to
light sources with CCT=4100 K slightly in contrast with the curves
presented in Figure 15. Pupil responses have also been noticed to
be larger on exposure of the nasal part of the retina (temporal
visual field) [112], having similar spatial characteristics as
melatonin suppression (as later noticed) [150].
Figure 15. (A) Mean spectral sensitivity curve for the photopic
pupil response (max 550 nm) of two subjects. Differential threshold
measurements () are plotted for 2 sec flashes of a 2 test patch
centrally fixated and seen against a continuous blue background.
Interrupted line, CIE photopic luminosity curve; solid line, mean
results of psychophysical measurements of photopic luminosity
(flicker photometry) on the same two subjects with the same
apparatus. (B) Solid line-Deviations of the pupil results under
scotopic conditions (max 500 nm) from the CIE spectral sensitivity
data (25 V criterion) and corrected for the absorption in the eye
media (double passage); , the b-wave of the ERG of the dark-adapted
eye. Mean results from two observers [105].
(A) (B)
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Circadian photobiology 17
2.3.3 Eye movements
There are four basic types of eye movements: saccades, smooth
pursuit movements, vergence movements, and vestibule-ocular
movements. They all have their own controlling neural circuitry.
Eye movements are very important as high visual acuity is
restricted only to fovea, and eye is always trying to direct fovea
to new objects of interest (foveation). Russian physiologist Alfred
Yarbus demonstrated in his experiments in the 1960s the pattern of
eye movements while examining an object [113]. Yarbus used contact
lenses with small mirrors attached to them to track eye movements.
Results can be seen in Figure 17 revealing subjects gaze while
viewing a bust of Queen Nefertiti. Thin lines represent the quick,
ballistic movements (saccades) and the denser spots represent
points of fixation where the observer paused to take in visual
information (only a few tens of milliseconds). First types of eye
movements, saccades, are rapid, ballistic movements that abruptly
change the point of fixation. The amplitude of saccades can range
from small correction movements (with reading) to larger (gazing
around a room) movements. The rapid eye movements during REM-sleep
are also saccades. Time behaviors of saccades are illustrated in
Figure 16, which shows that there is about 200ms delay if an
already fixated target starts to move. This delay is used to
compute an appropriate correction and if the target keeps moving,
another computation is needed. This is the main problem with
saccades as both the amplitude (how far) and the direction of the
movement should be computed as accurate as possibly. The amplitude
of the movement is controlled by firing duration of lower motor
neurons of the oculomotor nuclei. Figure 18 shows the control of
horizontal movement using lateral and medial muscles. The direction
of the movement is determined by which eye muscles are activated.
In principle any given direction could be controlled just summing
different eye muscle activity, but in reality the complexity of
such mechanism would be great. Basically the control has been
divided into two gaze centers: paramedine pontine reticular
formation (PPRF) or a horizontal gaze center; and rostral
interstitial nucleus or vertical gaze center. Centers can be
separately activated and the rotational movements are determined by
relative contribution of each center.
Figure 17. The eye movements of a subject viewing a picture of
Queen Nefertiti. The bust on the left is what the subject saw; the
diagram on the right shows the subject's eye movements over a
2-minute viewing period [39].
Figure 16. The metrics of a saccadic eye movement. The red line
indicates the position of a fixation target and the blue line the
position of the fovea. When the target moves suddenly to the right,
there is a delay of about 200 ms before the eye begins to move to
the new target position [39].
Figure 18. Motor neuron activity in relation to saccadic eye
movements. The experimental setup is shown on the right. In this
example, an abducens lower motor neuron fires a burst of activity
(upper trace) that precedes and extends throughout the movement
(solid line). An increase in the tonic level of firing is
associated with more lateral displacement of the eye [39].
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Circadian photobiology 18
Computation of the movements does not take place at gaze centers
as they get their input from the superior colliculus of the
midbrain and region called frontal eye field (Brodmanns area 8) as
seen in Figure 19. Both areas respond to visual stimuli and have
specific visual and motor maps equivalent to retinotopic mapping.
The responses of superior colliculus are better known than frontal
eye field. The simplified relation between superior colliculus and
frontal eye field is the following: the frontal eye field projects
to the superior colliculus and the superior colliculus projects to
the PPRF on the contralateral side (Figure 19), as it does also to
vertical gaze center which is excluded from the picture for the
sake of clarity. The frontal eye field then controls the eye
movements by activating selected populations of superior colliculus
neurons. It can also project directly to PPRF and control eye
movements independently of the superior colliculus. Frontal eye
field is also responsible for systematic scanning of visual field
to locate an object of interest from background noise. It was
thought in early 1970s when the collicular maps were found that
saccadic movements could be easily estimated using visual/motor map
matching. However later it has been found that saccade movements
dont necessarily even need visual stimuli. As seen in Figure 20,
nonvisual stimuli like auditory or somatic stimuli can activate
motor neurons and produce saccade movements. Also it has been
discovered that animals can be trained not to make saccades when an
object appeared to visual field, which led to a development of more
complex models as seen in Figure 20. We can see that theres a
direct connection between motor and visual neurons, which probably
provide the substance for the very short latency (~100ms)
reflex-like express saccades, which have been notices even after
the destruction of the frontal eye fields. The second type of eye
movement, smooth pursuit movements are much slower tracking
movements designed to keep a moving stimulus on the fovea. Smooth
pursuing movements are under voluntary control as person can decide
whether to follow some object or not. However only highly trained
individuals can make smooth pursuing movement without actual moving
target to follow, in reality most people just end up making a
saccade. Traditionally these movements were tested placing a
subject inside a rotating cylinder with vertical stripes, but
nowadays same test can be done using a screen with series of
horizontally moving vertical stripes. The eyes follow the stripe
end of their excursion followed by a quick saccade to opposite
direction for a pursuit of new stripe. This kind of mixed fast and
slow movement of the eyes is called optokinetic nystagmus. This is
illustrated in Figure 22, where after a quick saccade eyes are able
to follow the moving target smoothly. Vergence movements align the
fovea of each eye with targets located at different distances from
the
Figure 19. The relationship of the frontal eye field in the
right cerebral hemisphere (Brodmann's area 8) to the superior
colliculus and the horizontal gaze center (PPRF) [39].
Figure 20. The superior colliculus receives visual input from
the retina and sends a command signal to the gaze centers to
initiate a saccade. The terminals of the visual neuron are located
in the same region as the dendrites of the motor neuron [39].
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Circadian photobiology 19
observer. But unlike other eye movements, vergence movements are
disconjugate (or disjunctive) meaning that eyes move to opposite
directions, converging for close objects and diverging for far
objects. Convergence caused by near-field stimuli (or near reflex
triad) involves also pupillary constriction to increase depth of
field. Vergence movements are the slowest speed eye movement
although latency being less than with saccades. They are also very
small in amplitude, typically a few degrees. The last types of eye
movements, vestibulo-ocular movements, mean the compensation of
eyes to movement of head. When tilting your head you notice that
your fixating point remains more or
less at same point of your retina. The name vestibulo comes from
vestibular system, which main element is vestibular nuclei that is
situated in inner ear acting as accelerometer and spatial position
guide. The system extends through a large part of the brainstem;
simple clinical tests like the vestibulo-ocular response can be
used to determine brainstem involvement and possible damages, even
on comatose patients. The vestibular system detects brief,
transient changes in head position and produces rapid corrective
eye movements. However it is relatively insensitive for slow
changes. For example if the vestibulo-ocular reflex is tested with
continuous rotation and without visual cues about the movement of
the image (i.e., eyes closed), the compensatory eye movements cease
after only about 30 seconds. A person with vestibular damage finds
it difficult or impossible to fixate on visual targets while the
head is moving, a condition called oscillopsia (bouncing
vision).
2.4 LIGHT CHARACTERISTICS In this chapter the light
characteristics linked to the novel photoreceptor are reviewed in
regard to human circadian rhythms. The basic understanding of this
chapter is essential in designing the measurement equipment for
light exposure.
2.4.1 Spectrum
The peak wavelength of circadian responses is shifted towards
the blue end of the spectrum compared to the traditional visual
spectral sensitivities for photopic (V(), max=555nm), mesopic (max
between photopic and scotopic peak wavelengths) and scotopic (V(),
max=508nm) vision. According to current knowledge, the peak
wavelength seems to be around 480 nm [2] for ipRGCs. A series of
action spectra are presented in Table 1 concurrent with the
discovery of melanopsin (Provencio et al. [114], 1998) and ipRGCs
(Berson et al. [1], 2002).
Figure 22. The metrics of smooth pursuit eye movements. These
traces show eye movements (blue lines) tracking a stimulus moving
at three different velocities (red lines). After a quick saccade to
capture the target, the eye movement attains a velocity that
matches the velocity of the target [39].
Figure 21. Vestibulo-ocular eye movement (slow) resulting from
head rotation. This slow component is also called physiological
nystagmus, Fast eye movement are saccades that reset the eye
position [39].
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Circadian photobiology 20
Table 1. Analytic action spectra for circadian, ipRGC, and
ocular responses (modified from Brainard, 2006 [85]).
Species Biological responses Stimuli tested Peak [nm]
First Author Year
Human (Homo sapiens) Plasma melatonin suppression
8 fluence-response curves (hbw 10-15nm)
Est. max=464 (446-477)
Brainard [115] 2001
Human (Homo sapiens) Plasma melatonin suppression
6 fluence-response curves (hbw 5-13nm)
Est. max=480 (457-462)
Thapan [116] 2001
Mouse (Mus musculus) Pupillary light reflexes 6 fluence-response
curves (+/+) (hbw 10nm) (rd/rd cl)
Est. max=480 or 508
Lucas [117] 2001
Human (Homo sapiens) Cone cell ERG-wave 7 fluence-response
curves (hbw 10nm) Est. max=479
Hankins [118] 2002
Rat (Rattus norvegicus) ipRGC cellular depolarization
6/10 fluence-response curves (hbw 10nm) Est. max=484
Berson [1] 2002
Mouse (Mus musculus) Circadian phase shift 7 fluence-response
curves (rd/rd cl) (hbw 10nm) Est. max=481
Hattar [119] 2003
Mouse (Mus musculus), purified mouse melanopsin in vitro
Melanopsin-catalyzed GTP--35S uptake
Single irradiances of 4 restricted bandwidths (hbw 10-30nm)
Est. max=424 (420-44)
Newman [84] 2003
Monkey (Macaque nemestrina)
ipRGC cellular depolarization
10 fluence-response curves (hbw 15-20nm) Est. max=482
Dacey [2] 2005
Wild-type and retinally degenerate strains are indicated by
(+/+) and (rd/rd cl). Est. = Estimated max from fitting data to
spectral sensitivity curves or to visual photopigment nomograms.
hbw =half-bandwidth (hbw smaller than 10nm is considered
monochromatic).
The recent range for max of circadian responses has been from
459 to 484 nm with the clear exception of 420 nm by Newman et al.
[84], which study was done in vitro conditions and does not
necessarily represent the in vivo behavior of melanopsin as already
reviewed with melanopsin. Neither the study by Lucas et al. [117]
does not identify a max in the blue part of the spectrum in
pupillary responses of wild-type (+/+) mice, which was also found
in earlier studies [120-122] for phase shifting locomotor activity.
It could be that the intact rodent retina combines input from
ipRGCs and classic visual photoreceptors (cones more likely) for
phase shifting and pupillary responses. In contrast when mice do
not have functional cones or rods, their retinal sensitivity appear
to shift towards shorter wavelengths [119,122,123]. In photometry,
Abney's Law for additivity [124] has been used as a hypothesis for
the linear behavior of luminance perception. Additivity means that
the total luminance of a non-monochromatic light is the sum of the
weighted spectral radiations of the component wavelengths. However,
additivity does not hold for all lighting conditions. Additivity
failures occur both in photopic and mesopic vision [125], also
referred as the Abney Effect [126], that recognizes the failure of
the basic law. To make things even more complex this Abneys Effect
is known to be in error also [127]. In photopic vision, additivity
failure called sub-additivity occurs when the perceived brightness
is less than the sum of the component perceived brightnesses. This
phenomenon is apparently due mainly to non-linear cone-cone
interactions and is also called the Helmholtz-Kohlrausch effect
[128]. For example mixing monochromatic red light with
monochromatic green light of equal brightness can be seen less
bright than either of the two lights alone [129]. In mesopic vision
only the magnocellular channel appears to obey Abney's law of
additivity [130]. The first research by Figueiro et al. [131]
studying the circadian spectral opponency in humans compared the
melatonin suppression effects of blue light emitting diodes (LEDs)
and clear mercury (Hg) vapor lamps. Blue LEDs produced an
illuminance of 18 lx (29
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Circadian photobiology 21
W/cm2) at subjects eyes when the 175 W Hg lamp produced an
illuminance of 450 lx (170 W/cm2) at the eye. The radiant power of
polychromatic Hg lamp was set to produce at least equal or higher
melatonin suppression than the blue LED if additivity was to exist
in circadian response, following the univariance principle [132].
Results revealed a statistically significant difference between the
LED and Hg lighting conditions, with the LED condition resulting
stronger melatonin suppression in contrast to the theory of
additivity. The best-fitting function from the results is shown
Figure 23 which are relatively close match to the empirical action
spectrum for melatonin suppression by Brainard et al. [225]
(r2=0,86) and by Thapan et al. [116] (r2=0,84) both from 2001. In
conclusion the larger melatonin suppression by photopically
less-powered LED would indicate that spectral opponency exists in
human circadian system, and the results from studies done with
monochromatic light sources could not be generalized to normal
polychromatic sources used in architectural lighting. The first
circadian phototransduction model to incorporate the suggested
spectral opponency [131] was presented by Rea et al. [133] in 2005.
Compared to the previous models [91,134,356], it is much more
ambitious while still maintaining relatively simple mathematical
format. It is not limited on modeling the ipRGCs or melanopsin, but
it incorporates the basic mechanisms of other retinal neurons
involved in circadian phototransduction, as cones and rods are also
been proposed to be involved in circadian responses [119,135].
However, while the model is based upon a synthesis of a wide range
of existing literature in neuroanatomy, electrophysiology, and
psychophysics, main emphasis is on the results got on melatonin
suppression. Rea et al. [133] admit that this model is highly
likely to be changed as it lacks the more advanced features of
circadian phototransduction, but it still is a large step towards
more realistic models. Figure 24 shows the action spectra of the
proposed model. The proposed model [133] was later tested by the
same authors [136] with the results showing a relatively good fit
to the proposed model when tested with two polychromatic light
sources. The conventional views has been after the discovery of
ipRGCs that all non-image forming (NIF) functions have the same
action spectra as it for example found that short-wavelength light
(460 nm) is more effective in alertness-promoting than light at
550nm [137-139], but a recent study by Revell et al. [140] revealed
that light at 420 nm was more effective in alertness-promoting than
light at 470 nm. This would mean that the action spectrum presented
for melatonin suppression [116,133,225] would not be accurate for
alertness promotion. This could mean that human melanopsin could be
really most sensitive to short wavelengths at 420-430
Figure 23. Hypothetical opponent action spectrum for melanopsin
consistent with the present [131] and previous results [116,225].
Curve from Figuiero et al. [131].
Figure 24. Predictions of the model to the constant criterion
spectral sensitivity data of Brainard et al. [225] and of Thapan et
al. [116]. Graph by Real et al. [133].
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Circadian photobiology 22
nm as shown in some studies [83,84]. However, this peak
wavelength of 420-430 nm is in contrast with the other data from
melanopsin action spectrum [2,81,82], cone ERG [118], and circadian
phase shifting [141]. Human eye undergoes age-related changes in
total and in wavelength-dependent transmittance. It would be
natural to assume that these changes would have some kind of impact
to circadian phototransduction as well. As in both early studies by
Brainard et al. [225] and Thapan et al. [116] the action spectra
were corrected for absorption in the lens (as authors wanted to
obtain an action spectrum that applies to the irradiance level at
the retina), it is reasonable to study what are the real
differences in circadian responses due to properties of cornea,
aqueous, lens and vitreous, which light has to pass before reaching
the retina. As noticed already in Figure 11, corneal transmittance
is relatively constant between the range of 400 nm to 600 nm and
above all the corneal transmittance does not differ significantly
as a function of age at any wavelength [100]. Although absorption
and scatter in humours may have minor effects, the most relevant
part of the human eye is the crystalline lens as it has been noted
to yellow with age thus attenuating short-wavelength light. Another
significant age-related change in ophthalmologic optics is the
senile miosis [142], where pupil diameter changes with age under
both light-adapted (diameter decreases) and dark-adapted (diameter
increases) conditions. The relative pupil area has its maximum
value at the age of about 15 years and is reduced throughout adult
[99], and it this factor alone reduces the retinal illuminance to
half in the eye of a 70-year-old. The following equation can be
used to calculate the relative efficiency of light for suppression
of melatonin with regard to the age according to Charman et al.
[99]:
= d )S(A)(T)(T)(ER LC (4) Where, R = effective irradiance at the
retina
E() = spectral irradiance of the source at the cornea TC() =
transmission of the cornea (near-axial path) TL() = transmission of
the lens (near-axial path) A = pupil diameter (near-axial path) S()
= melatonin action spectrum
According to this formula, the efficiency of light should
decrease with increasing age; however an experimental study failed
to verify this assumption [143] and concluded that there is no
correlation between efficiency of melatonin suppression and age of
the subjects. No gender-related differences in melatonin
suppression have been yet discovered [144]. The values of TC(),
TL() and A are measured when light enters the eye along a
near-axial path assuming that no effects of the Stiles-Crawford
type [145,146] will occur, as the ganglion cells lie anterior to
the outer segments of the receptors which are responsible for any
waveguiding effects.
2.4.2 Spatial distribution
Relatively little is known about the spatial distribution of
melanopsin-containing ganglion cells (ipRGCs) in human eye. This
knowledge is important in knowing where to place the lights in
order to produce the maximal biological responses. The results from
various studies [147-150] indicate that a significant gradient in
density of melanopsin-containing retinal ganglion cells is present
both in the horizontal and in the vertical direction. The highest
density of melanopsin-containing retinal ganglion cells (ipRGCs)
would seem to occur in the inferior nasal area of the retina
corresponding to upper (superior) temporal (lateral) visual field.
The ratio between temporal retina and nasal retina for
melatonin
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Circadian photobiology 23
suppression was 0.54 in the study by Visser et al. [147] and
0.59 in the study by Rger et al. [150]. The difference was even
larger between upper (superior) and lower (inferior) retina in the
study by Glickman et al. [149] where melatonin suppression was
~6,3% for upper retina whereas it was ~29,1% after a 90 minute 200
lx polychromatic light exposure (percentages plotted from the
graphs). This would indicate a ratio of 0.22 between upper and
lower ratio.
2.4.3 Intensity
Despite of the relatively large amount of studies on circadian
phototransduction, only a few systematic studies [157,192] have
been done on the influence of light intensity on the phase shifting
and melatonin suppression. The early studies done with subjects
which allowed to self-select their sleep-wake cycle showed that
only bright light could affect human circadian rhythms [151,152],
one study reporting a threshold of 1500 lx [153] and the others
showed a significant phase shift with illuminances as high as 4000
lx [154] and 5000 lx [155]. In the human study by Boivin et al.
[156] the phase resetting response was reported to increase with
light intensity in a nonlinear manner. In the study by Zeitzer et
al. [157] the intensity response curve (IRC) between illuminance
and the phase resetting response was also found to be nonlinear.
This found nonlinearity is consistent with a cube-root compression
of illuminance as a function of the illuminance and (phase
resetting) response, reported previously for visual perception
[158]. In non-human mammals, the intensity dependence of both phase
shifting of the circadian pacemaker and acute suppression of
melatonin have been well characterized [159-161]. In general, the
results obtained by Zeitzer et al. [157] are the most commonly used
as a reference for the light intensity required for melatonin phase
shift (Figure 25A) and melatonin suppression (Figure 25B). As
little as ~100 lx of (corneal) light could produce half of the
maximal phase delay shift found at 10000 lx and that 90% of the
asymptotic maximum response could be achieved with 550 lx. This
would indicate that human circadian pacemaker is highly sensitive
to ordinary room light and that minor changes in room light
intensity could have a major impact on entrainment of the human
circadian pacemaker. This is not consistent with some previous
studies [154,162,163] which failed to
Figure 25. Illuminanceresponse curve of the human circadian
pacemaker. The shift in the phase of the melatonin rhythm (A), as
assessed on the day following exposure to a 65 h experimental light
stimulus, has been fitted with a four parameter logistic model
using a nonlinear least squares analysis. Acute suppression of
plasma melatonin (B) during the light exposure also has been fitted
with a four parameter logistic model using a nonlinear least
squares analysis. The logistic models predict an inflection point
of the curve (i.e. the sensitivity of the system) at 120 lx.
Saturation of the phaseshift response is predicted to occur with
550 lx and saturation of the melatoninsuppression response is
predicted to occur with 200 lx. Individual subjects are represented
by , the model by the continuous line, and the 95% confidence
intervals by the dotted lines [157].
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Circadian photobiology 24
find significant phase resetting with room light but is however
supported by several studies [164-166,192] with similar results.
However, it should be noticed that there seems to be maximal
melatonin suppression rate that is independent of the light
intensity used [167,168]. Results of McIntyre et al. show
approximately a rate of 1,5% per minute of light exposure until
reaching an asymptotic level between 30 and 60 min. Similar
observations have been made from animal studies [169-171]. A
comparable is found in electronics and known as the slew rate,
whereby the output of an amplifier cannot keep up with rapid
changes in the input. Maximum nocturnal melatonin suppression would
be about 45-50% after 30 minute of bright light. The accurate
measurement of retinal illuminance is more difficult than measuring
horizontal task illuminance as retinal illuminance depends on the
angle of gaze, position of the head, pupil size [172], lens
transmission [99], and possible photophobic response such as
squinting [173-175]. For example, Sliney [175] estimated that
squinting results in a log unit reduction in retinal illuminance
compared to the estimated retinal illuminance using photometric and
pupillometric measurements. In practice this means that higher
corneal illuminance can produce smaller melatonin suppression than
lower corneal illuminance even though pupil size is measured
continuously as occurred in a study by Figueiro et al. [136]. An
example of the relation between photopic illuminance at cornea can
be seen in Table 2 from the study by Figueiro et al. [136], where
it can be seen that light source with a CCT (correlated color
temperature) of 8000K produced larger melatonin suppression with
1000 lx than with 300 lx. It can be also seen that as corneal
illuminance increased the pupil size decreased, and the mean pupil
are area was smaller with light source with higher CCT.
Table 2. Corneal irradiance, mean pupil area, retinal
illuminance value and mean melatonin suppression (meanS.E.M.) for
each lighting condition [136]. Light source
Photopic illuminance at cornea (lx)
Irradiance at cornea (W/cm2)
Mean pupil area (mm2)
Retinal illuminance (lx mm2)
Mean melatonin suppression (%)
30 8.2 19 573 -3% (11%) 100 27 12 1150 10% (4%) 300 82 8.9 2670
38% (7%) 4100 K 1000 270 5.8 5800 38% (6%)
30 9.7 16 492 10% (8%) 100 32 10 1010 32% (7%) 300 97 8.2 2460
47% (4%) 8000 K 1000 320 5.0 5000 34% (9%)
2.4.4 Timing
The amount by which a discrete light pulse can change the timing
of the circadian system is phase dependent, and this phase
dependency is described by phase response curves (PRC). In general,
there are two general PRC morphologies: a low amplitude PRC with
maximal phase shifts of a few hours (Type 1), and a high amplitude
PRC with phase shifts as large as 12 h (Type 0) [176,177]. In Type
0 resetting, the resetting stimulus affects both the phase and
amplitude, and a stimulus of appropriate strength applied at a
critical phase can in theory reduce the amplitude of oscillation in
zero (singularity) [177-179]. Single bright light elicits phase
shifts in humans consistent with Type 1 PRC [180,181] showing
typically phase advances of ~2 h and maximum phase delays of ~3 h.
In both the Type 1 and Type 0, phase shifts in response to light
are observed during the biological night when humans are habitually
asleep in the dark. A recent study by Khalsa et al. (2003) [182]
was the most comprehensive study on human PRC so far. In this study
a 9 day in-laboratory study protocol was used preceded by 2 weeks
of regular 8 h sleep schedule based upon subjects (n=43) habitual
sleep and wake times. The results [182] supported previous findings
[183,184] that there is no dead zone
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Circadian photobiology 25
(when no phase shift is elicited by bright light) in the human
PRC. Three different PRCs from the study can be seen in Figure 26,
which differ by the phase markers used. Figure 26A uses melatonin
midpoint, Figure 26B dim light melatonin onset (DLMOn), and Figure
26C dim light melatonin offset (DLMOff) as the phase marker for
circadian rhythm (for phase marker details see Figure 3). The
transition from delays to advances in the critical region (CT 0) is
rapid, while the transition from phase advances to phase delays
during the subjective day is more gradual. The phase shifts
measured by DLMOff are smaller than those measured by DLMOn [182],
which is consistent with the results obtained from rodent studies.
It has been hypothesized that there may be two coupled oscillators,
an evening or E oscillator associated with melatonin onset, and a
morning or M oscillator associated with melatonin offset
[185-188].
2.4.5 Duration
Traditionally bright light experiments have consisted of 2 to 8
hour continuous exposures [189-192], based on the assumption that
bright light exposure is consistent with the Bunsen-Roscoe law that
states that the effect is independent (within a certain general
time frame) of the duration of exposure as long as the radiant
exposure is the same [91,193]. However evidence from animal
experiments [194-196] would suggest that the same phase-shifting
than with continuous exposure could be achieved with intermittent
light exposure with less radiant energy. The response of human
circadian system has not been very well quantified even though the
exposure to bright light is typically intermittent in everyday life
[197-200]. Kronauer et al. [201,202] have proposed a revisal model
for the resetting effect of light. The model is partly based on
experiments comparing the effects of continuous and intermittent
bright light stimuli (~9 500 lx) over a ~5-h period. The results of
a study by Rimmer et al. [203], designed to test the model of
Kronauer et al. [201,202], suggest that an intermittent bright
light stimulus, interrupted by intervals of complete darkness that
exceed the light exposure can significantly phase shift the human
circadian pacemaker. When bright light occupied only 31% of the
total stimulus, 70% of the median resetting response was observed.
Furthermone, when bright light occupied 63% of the total stimulus,
nearly 90% of the median resetting response was preserved. These
findings also indicate that the brief intermittent exposures to
bright light that are normally encountered in everyday life (during
the night and day) [197,199,200] may have a greater impact on
circadian entrainment than was previously recognized
[198-200,204,205]. Studies by Boivin and James [206], by Baehr
[207], and Gronfier et al. [208] also support this
Figure 26. Phase advances (positive values) and delays (negative
values) are plotted against the timing of the centre of the light
exposure relative to the melatonin on the pre-stimulus CR (defined
to be 22 h), with the core body temperature minimum assumed to
occur 2 h later at 0 h. Using A) melatonin midpoint, B) dim light
melatonin onset (DLMOn), and C) dim light melatonin offset (DLMOff)
as marker for circadian phase. Data points from circadian phases
618 are double plotted. The filled circles represent data from
plasma melatonin, and the open circle represents data from salivary
melatonin in one subject. The solid curve is a dual harmonic
function fitted through all of the data points. The horizontal
dashed line represents the anticipated 0.54 h average delay drift
of the pacemaker between the pre- and post-stimulus phase
assessments. The fitted peak-to-trough amplitude of the DLMOn PRC
(5.41 h) appears slightly larger than that of the DLMOff PRC (4.60
h) [182].
C B A
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Circadian photobiology 26
proposed model [201,202] indicating that sustained periods of
intensely bright light are not necessary for resetting the human
circadian system.
2.4.6 Photic history
It has been shown that resetting response of circadian pacemaker
can be attenuated by a preceding nonsaturating stimulus in animals
[209], and phase shifting in mammalian has been shown to be maximal
after prolonged exposure to complete darkness before a stimulus
[210,211]. Also a study by Hebert et al. [212] made in humans
showed significant differences (with large inter-individual
variability) differences in melatonin suppression after different
(dim vs. bright) light history. These studies would suggest that
the light-mediated melatonin suppression could also be modulated by
prior photic history. Study by Smith et al. [213] revealed a
significant difference in melatonin suppression between two
lighting history conditions, with a mean suppression of 71,2%
(7,1%) in the approximately 200 lx prior light history condition
vs. a mean suppression of 85,7% (6,5%) in the approximately 0,5 lx
prior light history condition. The results [213] demonstrate that a
prior light history alters light-mediated melatonin suppression,
while it is impossible to determine whether dim background
potentiates or relatively bright background diminishes the
strength. It would seem important always to control the prior light
history when examining melatonin suppression. Findings suggest that
a controlled photic history of 63 hours before a light stimulus is
sufficient to change the suppression effect of the subsequent light
stimulus. However it is not possible to determine the exact time
from this study [213] to be a sufficient control time and further
investigation is needed for more quantitative results.
2.4.7 Polarization
The differences between nonpolarized and vertically polarized
light to melatonin suppression were investigated by Brainard et al.
[214] in 2000. Six subjects participated in the study and they were
exposed to four different light intensities: 20, 40, 80 and 3200 lx
(for saturation response) with their pupils dilated with
cyclopentolate HCl. The results of the study [214] revealed hat
there is a significant correlation between the light intensity used
and melatonin suppression, but no significant differences between
nonpolarized and polarized light.
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Eye and photometric measurements 27
3. EYE AND PHOTOMETRIC MEASUREMENTS
Literature review is done on the available methods to measure
eye movement, pupil size and light entering the eye for proper
quantification of circadian effective light exposure. Also the
electrical activity recordings of the eye ERG and/or EOG are
reviewed as they can be used for further quantification of
physiological responses of light. Even though within the scope of
this work they are not integrated to the dosimeter device but the
possible recording of EOG/ERG should be taken account when
designing the dosimeter.
3.1 ELECTRICAL ACTIVITY Two the most typical measurements of the
electrical activity of the eye are electroretinogram (ERG) and
electrooculogram (EOG). ERG is more clinical utility used for the
diagnostics of various eye diseases but it can also be used to
measure circadian responses as done by Hankins et al. [215]. EOG is
commonly used in studies measuring changes in alertness as well as
in EEG (electroencephalography) studies for the elimination of the
eye blink artifacts in EEG recordings.
3.1.1 Electroretinogram (ERG) Electroretinogram (ERG) is a
device measuring the electrical activity of the eye. Figure 27A
[216] shows schematically the basic measurement setting with a
special saline filled contact lens with an Ag/AgCl electrode placed
on top of cornea As it is shown in Figure 27B [216], light pulse
induces a potential change and four common ERG waveforms (the a, b,
c, and d waves) are marked to the picture. It should be noted that
notations can differ in literature.
The first (a) component is early-receptor potential (ERP), which
appears almost instantaneously after onset of light. The amplitude
of the ERP depends directly upon stimulus intensity and the
concentration of visual pigment in the outer segments of the
photoreceptors. Therefore, the ERP is believed to reflect dipole
changes in the visual pigment molecules due to conformational
changes that are elicited by photon absorption. The ERP has been
used in research to follow non-invasively the concentration of the
visual pigment during light adaptation and in the dark following an
exposure to bright light that causes substantial pigment bleaching
[217]. This is followed by (b) late-receptor potential (LRP), which
has a small latency (1-5ms) and is found to be maximal near the
synaptic endings of the photoreceptors therefore
Figure 27. (A) The transparent contact lens contains one
electrode, shown here on horizontal section of the right eye.
Reference electrode is placed on the right temple. (B) Typical
vertebrate ERG waveform in response to a 2 s light flash [216].
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Eye and photometric measurements 28
reflecting the outputs of the photoreceptors. ERG and b waves
can be used to study the diurnal variation in the cone pathway
[218] which is related to the diurnal transition between processes
optimized for high (photopic) and low (scotopic) light levels
[219-222]. Cone b wave-implicit time appears to be regulated by
environmental irradiance as an adaptation to the varying demands of
the solar cycle [222]. This regulation seems to be driven by the
novel photoreceptor (ipRGC), and by studying the irradiance and
wavelength dependent reduction in b wave-implicit time it is
possible to study the spectral sensitivity of the novel
photoreceptor as done by Hankins et al. [223]. Even though ERG has
not been used in further studies examining the non-image forming
(NIF) responses in humans, it could be added to some experimental
designs to provide supplemental information in addition to typical
measures (e.g. melatonin, CBT). For example Hankins et al. [223]
controlled retinal illumination by using a custom-built Ganzfeld
dome illuminator [224] (similar apparatus as Goldman perimeter
[225,226]) with the ERG electrodes attached bilaterally beneath
each eyelid with a forehead reference ground [222]. The c-wave is
now known to originate in the pigment epithelium after the
discovery of potassiumretinogram (KRG) [227]. C-wave is also called
'The standing potential of the eye'. Although the c-wave originates
from the pigment epithelium, it depends upon the integrity of the
photoreceptors, because light absorption in the photoreceptors
triggers the chain of events leading to the decrease in
extracellular concentration of potassium ions. Therefore, the ERG
c-wave can be used to assess the functional integrity of the
photoreceptors, the pigment epithelial cells and the interactions
between them. The d-wave is only evident when the ON and OFF phases
of the ERG response are separated in time, by using light stimuli
with long duration (>100ms). With shorter durations d-wave tends
to be combined with the b-wave. ERG measurements can be also used
to determine the perception of flickering lights as with 100 Hz
fluorescent lamp flicker [228,229]. Even though no visual
perception of flicker exist the response to flicker can be seen in
ERG, and this flicker perception will most likely cause the
problems associated with clearly visible flicker such as headaches
and fatigue. Also cone and rod ERG responses can be isolated using
different (colored) stimuli as seen in Figure 28A [231]. Rods are
also incapable of following long light flicker (not really evident
in short timescale of Figure 28A [231]), and it is possible to
determine the involvement of rods and cones.
(A)
(B)
Figure 28. (A) Cone and rod ERGs can be isolated using dim flash
stimuli into photopic (cone) and scotopic (rod) signals [231]. (B)
Using different rates (flicker) of stimulus presentation also
allows rod and cone contributions to the ERG to be separated. Even
under ideal conditions rods cannot follow a flickering light up to
20 per second whereas cones can easily follow a 30 Hz flicker,
which is the rate routinely used to test if a retina has good cone
physiology [231].
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Eye and photometric measurements 29
3.1.2 Electooculogram (EOG) The electrooculogram measures the
potential that exists between the cornea and Bruch's membrane at
the back of the eye. The potential produces a dipole field with the
cornea approximately 5 millivolts positive compared to the back of
the eye, in a normally illuminated room. Although the origin of the
EOG is the pigment epithelium of the retina, the light rise of the
potential requires both a normal pigment epithelium and normal
mid-retinal function. Elwin Marg named the electrooculogram in 1951
and Geoffrey Arden [230] developed the first clinical application
[231]. It far less invasive method compared to ERG as seen in
Figure 30 [231]. The electrodes of the EOG are normally placed at
the outer canthi of each eye, one slightly above the cantomeatal
place, the other slightly below [232]. EOG is also the abbreviation
for electro-olfactogram, which is used to determine electrical
responses of different smells and scents, and thus is totally
different device [233]. EOG is frequently the method of choice for
recording eye movements in sleep and dream research [234], in
recording eye movements from infants and children, and in
evaluating reading ability and visual fatigue. And the most
important application within this work is its use in slow eye
movement (SEM) measurement with EEG to assess alertness. Figure 29
[231] shows a 10-second periods of eye movement back and forth
between two red LED lights placed 30 degrees apart inside a
Ganzfeld measurement device. After training the patient in the eye
movements, the lights are turned off. About every minute a sample
of eye movement is taken as the patient is asked to look back and
forth between the two lights. As various types of eyelid and eye
movement patterns have been shown to respond to sleep loss and to
correlate with sleepiness in a variety of protocols [235-237]
indicating that EOG could be in theory used to assess sleepiness
objectively [238]. However, in practice the EOG recordings have
shown too large inter-individual differences making objective
alertness assessment still a thing of the future [239-241].
Aserinsky and Kleitman [242] described SEMs during drowsiness
preceding sleep onset and during light sleep. Kuhol and Lehmann
[243] found that SEMs became larger and more regularly sinusoidal
when simultaneous showing of the EEG was noted during sleep onset.
Slow (0,25 Hz), pendular, horizontal eye movements wer