What is macular pigment?
It is a yellow mixture of two carotenoid pigments, lutein and zeaxantin
This is lutein:

and this is zeaxanthin:

Note the difference between the two is the position of the double bond in the six carbon ring at the right, here shown in red.
These carotenoids are derived from plants we eat and they are concentrated in the eye.

The retina is the light sensitive layer of cells at the back of the eye.  It has a curious structure with light passing from the front of the eye and penetrating several layers of retinal nevre cells before it reached the photosensitive rods and cones.  The retina is said to be "inverted" because of this apparently back to front arrangement.  In the centre of the visual field where the visual acuity is best (the centre of gaze) the nerve cells overlaying the cone photoreceptors are displaced to one side making a kind of pit which is called the macula.   This structure give the best optical resolution without any interference from the other nerve cells.

The picture on the left is a scanning electron micrograph of a human retina showing the right hand side of the macula.  The superficial nerve cells of the inner retina are clearly seen to be displaced.  The position of the macular pigment is shown as a yellow haze beneath the inner retina but above the photosensitive portion of the photoreceptors which are the rod shaped sructures.

What does the pigment do?
It is a free radical scavenger.

• Free radicals are dangerous and highly reactive chemical entities that rapidly damage tissues and kill cells
• They are a by-product of normal metabolism
• Their production is enhanced by high oxygen concentrations and energetic radiation (short wavelength light)
• The macula has a high metabolic rate, a high oxygen tension and light is focused upon it by the cornea and lens
• The macula is thus vulnerable to attack by free radicals if they are not rendered harmless by the macular pigment
• The capacity of the pigment to nullify the effects of free radicals is limited

Why should we care?
After a life time’s exposure to free radicals, the photoreceptors (rods and cones) of the retina may decay prematurely  - a condition called AGE RELATED MACULAR DEGENERATION (ARMD).
Macular pigment may slow this process so the more macular pigment you have, the better protected your retina may be.

What should I do to increase my macular pigment?
Follow Popeye’s example: EAT YOUR GREENS

Some experts recommend a daily lutein intake of 6 mg from food although the average intake is probably a sixth of this.  As a guide, the table shows how much lutein and zeaxanthin (microgram per 100 gram) there is in a half-cup serving of the vegetable (selected values from an official site with all kinds of foods listed: http://www.nal.usda.gov/fnic/foodcomp/Data/car98/car_tble.pdf).   The figures vary greatly from sample to sample and are an approximate guide only.   The figure quoted is for lutein and zeaxanthin together. The general advice for a diet rich in lutein and zeaxanthin is to eat plenty of dark green vegetables.

How do I find out how much pigment I have?
Measurement of macular pigment can be done in vitro or in vivo.   In the former in vitro measurements the eyes are removed and dissection is followed by microscopic examination or by spectrophotometry directly on the tissues. Alternatively, the pigment can be extracted with solvents and chromatography, spectrophotometry or fluorescence spectrophotometry are used. Of course, none of these methods is much use to a living individual so in vivo techniques are employed.

In vivo techniques are all based on the idea that the macular pigment, because it is yellow, absorbs blue light and this happens in the macula.  Away from the macula, there is no macular pigment and the blue light is not absorbed (as much).  Several methods have been used over the years and a few are described here.

Fundus Reflection Densitometry


The top pair of schematics above show blue light and green light being reflected from the macula: the reflected blue light is attenuated because it is absorbed by the yellow macular pigment.  In the lower pair, the light is reflected from outside the macula and the reflected blue light is not attenuated.  To obtain an indication of how much macular pigment is present, all that is necessary is to collect the reflected light and in some way determine the ratio of blue to green in the macula and the parafovea.  This has been done in several ways:

TV Fundus Reflectometry
Kilbride, PE. et al, (Vision Res. 29:663, 1989) used a TV camera with special illumination at 462 nm (blue) and 559 nm (green) wavelengths of light to take pictures of the fovea.  They got a computer to digitise and then subtract the green picture from the blue picture and thus got an indication of how much macular pigment was present, and how it was distributed over the retina.  The picture below shows the blue picture.  Even before the green one is subtracted the darkening in the macula is apparent.

Kilbride's computer produced produced plots of light absorption as shown below and it is easy to see how
Blue - Green = Pigment
Both the blue and the green light are absorbed in about equal amounts by haemoglobin and melanin which are present throughout the retina.  Their absorption is cancelled out by this subtraction process which leaves a plot of just the macular pigment.

Scanning Laser Ophthalmoscopy
The introduction of the scanning laser ophthalmoscope has meant that it is possible to examine the retina when illuminated by laser light of closely specified wavelength (colour).  This principle has been used by a  number of workers including Tony Halfyard (Institute of Ophthalmology London, personal communication, 1999) who have done much the same as Kilbride et al  but with different imaging technology.   Halfyard used a blue-green laser and a red laser but these still can give a good picture of where the macular pigment is and how much is present.  The figure below shows the subtractive techniques he used to reveal the pigment and the concentration in the macula is obvious  (by the way, these pictures are of my retina).

Because he used a blue light of wavelength 488nm which is not at the peak of the spectral absorption curve of the macular pigment (460nm) it is necessary to correct the value for the amount of pigment present as shown below:

The correction factor is about  x 1.3 and is easily applied.

Other in vivo Objective Techniques

Other authors have used the SLO, e.g. Berendschot, v d Kraats & v Norren (Ophthalmic Research, 30/S1, Ever Meeting Abstracts, Karger, Basel, 1998) who compared SLO and spectral fundus reflectometry with a subjective method and found the former the best.
All objective methods are complex and are currently lab-based, often using mouth bites to position accurately the subject's head for Maxwellian view - this is where the light that enters the eye is focused into a small beam to pass through the pupil.  The optical arrangements are complex and expensive, e.g. monochrometers and xenon lamps.
Delori FC. et al (Invest Ophthalm Vis Sci. 36:718, 1995) measured the fluorescence of the melanin in a small spot in the macula and a few degrees outsdie it.  Again subtraction of one set of data from the other yielded a difference spectrum which is that of macular pigment.  The results are clearly useful, as shown here for two subjects:


From Delori FC. et al, Invest Ophthalm Vis Sci. 36:718, 1995

SUBJECTIVE TECHNIQUES
Instead of using objective measurement techniques, many authors have used subjective techniques where the observer has to make some judgement about the appearance of a test object.  This is really psychophysics and though such techniques are seldom as accurate or precise as objective techniques, they are often easier to use and cheaper to set up.  Some of the methods are:

Haidinger’s Polarisation Brushes
Bone RA, et al, Vision Res, 32:105, 1992
An intriguing application of an entoptic "illusion"

Anomaloscope
Moreland JD, et al, Vision Res, 38:3241, 1998
The anomaloscope is used to measure colour vision and here is used to detect and quantify the macular pigment 

Heterochromatic Flicker Photometry
Werner JS, Wootten BR. J opt Soc Amer, 69:422, 1979
This is the subjective technique that is now widely used: it is explained below

HETEROCHROMATIC FLICKER PHOTOMETRY
A small test field about one degree across alternates between blue and green light several times a second.  The colours are chosen, as in the objective techniques, so that the  wavelength of the blue light corresponds with the peak of absorption of the macular pigment and the wavelength of the green so that it is not absorbed by the pigment.   Usually the intensity of the green light is fixed but that of the blue is adjustable by the subject whose task is to reduce the appearance of flicker to a minimum.   When the perceived luminance of the blue and green lights are different the flicker is very obvious but when they are the same, the flicker is minimal.  The subject makes two sets of measurements, one with the test field imaged on the fovea and the other with the test field imaged in the parafovea where there is very little or no macular pigment.  The principle is shown below:

When minimum flicker is achieved in the two locations, the optical density of the macular pigment is:
   
    optical density of MP =log I_F - log I_P

where I_F is the luminance of the blue light for minimum flicker in the fovea and I_P is the luminance of the blue light for minimum flicker in the parafovea.

There are some caveats with heterochromatic flicker photometry and this list is taken from points made by several authors in recent publications:

• the match is made with M & L wavelength cones
• it is assumed that the ratio of M to L cones, & their sensitivity, is constant across the central retina: this is so (Nerger & Cicerone, Vis Res, 32:879, 1992)
• the S cones should play no part in the match: their influence may be reduced by
  1 choice of test wavelengths to which M & L cones are much more sensitive than S cones
  2 S cones are fatigued by a blue adapting background of a wavelength to which M & L cones are not very sensitive
  3 a flicker rate of 13 Hz is near the fusion frequency of S cones but well below that of M & L cones
• the influence of rods is reduced as 13 Hz is above fusion frequency of rods
• optimum flicker rates for obtaining good nulls at the luminance match varies from person to person and 13 Hz is a compromise
• the intensity of the constant green test field should be five to ten times the intensity of the blue background and in the photopic range
• macular pigment optical density (MPOD, a measure of the quantity of pigment present) depends on test field size as the null match is heavily influenced by the field edge
• null matches are easier to make in the parafovea so subjects should start there
• for some subjects the Troxler fading of the test fields can be a problem
• sometimes chromatic aberration can limit the ability to make null matches
• up to 5% of subjects cannot obtain a minimum flicker match
• MPOD should agree between eyes within 0.1 and repeats measures within about 0.08 log units

There have been many papers describing the use of heterochromatic flicker photometry (HCFP) to measure macular pigment.  Currently the best known group is working at Harvard - see Snodderly and Hammond (Chapter 13, In Vivo Psychophysical Assessment of Nutritional and Environmental Influences on Human Ocular Tissues: Lens and Macular Pigment, in Nutritional and Environmetnal Influences on Vision, ed. Allen Taylor, CRC Press, Boca Baton, 1999) for a good review of the topic.
Typically, various authors have employed optical laboratory techniques with arc lamps, monochromators and rotating shutter discs to present their subjects with the appropriate blue and green flashing lights.  The use of interference filters has helped to simplify the design of the apparatus, but it has usually been rather large and expensive.   With the suggestion that the onset of age-related macular degeneration might in some way be linked with the amount of macular pigment, which is thought to provide protection for the retina against oxidative stress (it is an antioxidant), it became important to be able to measure macular pigment in situations other than research laboratories.  A small, portable apparatus was required that could be taken into any clinic or other location.

At the University of Westminster, we had been using light emitting diodes (LEDs) in investigations of traffic signals and we knew something of their properties.  They seemed potential candidates for light sources for a macular pigment measuring instrument (we call such an instrument, rather inaccurately, a maculometer) because as light sources LED’s are:

• near monochromatic
• easy to drive with simple electronics
• small and rugged
• inexpensive
• do not require cooling.

We were able to select LED's of appropriate wavelengths which were readily available.  The figure beneath shows how well the radiation output of the LED's matches the absorption curve of the macular pigment (shown in grey).

We thought we could make a small portable instrument using LED's.  The first one we made was in a shoe box and we took the opportunity to redefine the overall concept - we did not want the subject constrained by a mouth bite, head band or chin rest so we arranged for the blue adapting background to be viewable around the blue/green flashing test field without the need for high precision alignment and imprisonment of the subject.   We did this by projecting a diffused image of an array of suitable LED's via a sheet of glass at 45 degrees into the space to be occupied by the subject's eye - see the diagram below.  In this way the subject was free to move their eye in an area about 30 mm across without changes in the appearance of the adapting field or the test field.   We also wanted a reliable instrument with no moving parts which are typical of other methods where a rotating shutter or similar device causes the test field to alternate between blue and green light.  With LED's it is possible to switch electronically from blue to green, thus avoiding mechanical components

A general view of the prototype (made in a shoe box) with the lid removed and its electronic box is shown below (right).  Also shown (left) is the subject's view of the adapting field and the test field (lid removed).

The red fixation LED is provided to make the minimum flicker matches on the parafovea: for the foveal matches, the subject looks straight at the test field.  To make the instrument yield reliable measurements, we had to tailor the properties of the electronic drive circuitry to the psychophysical behaviour of a typical subject.  This instrument was described at the European Association for Vision and Eye Research (EVER) meeting in Palma de Mallorca, October 1998 (Ophthalmic Research, 30/S1, Ever Meeting Abstracts, Karger, Basel, 1998).

The prototype was a success but it was considered fragile (the lens, glass plate, etc.) and expensive (lens).  Consequently the instrument was re-designed.  It went through a number of versions but at the EVER meeting in October 1999 (Ophthalmic Research, 31/S1, Ever Meeting Abstracts, Karger, Basel, 1999), again in Palma de Mallorca, we described the current instrument which dispenses with any optical components - no lens, no glass.  The three parts of the instrument are shown below - the subject views the test field and background through the aperture in the Optical Unit (left of picture) resting their forehead against the blue guide bar, adjusts the control on the Subject's Unit for a minimum flicker match and the operator reads of the setting from the meter on the Operator's Unit (picture right).

	On the left is shown the eyemet Maculometer which is currently being used in clinics in Europe on a range of trials

The need to change fixation to measure the parafoveal matches is eliminated by using an annular test field, as shown below (left).  The central foveal test field is illuminated only by a dim red LED to provide a fixation point for the subjects whilst they make the parafoveal matches with the annular field alternating blue and green  To make matches in the fovea, the annulus and the red fixation LED are turned off and the foveal test field flickers blue and green.  The two photographs (below right) show the subject's view of the foveal test field (upper) and the parafoveal test field and red fixation spot (lower).  The test fields are set to work at high luminances, around 150 cd.m^-2, so the measurements can be carried out successfully in rooms that are not very dim.  The picture below (centre) shows a subject making a match.

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