Chemistry Reference
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a digital format and analyzed using the Brindley and Willmer model, that is, the difference between
the log-transformed images was assumed to represent the (double) optical density distribution
of MP. The majority of MP rel ectometry studies have employed the scanning laser ophthalmoscope
(SLO) rather than a standard retinal camera (Elsner et al. 1998, 2000, Berendschot et al. 2000,
Wüstemeyer et al. 2002). While the SLO is a relatively expensive instrument, it is comparatively
immune to the problem of scattered light in the eye's optical media that can degrade the images.
Images of the bleached retina are typically captured at 488 and 514 nm, conveniently the wave-
lengths of an argon laser, and sufi ciently close to the wavelengths of peak and zero absorption of
MP. It is usually assumed that spatial variations in optical density of pigments in the light path other
than MP may be neglected and, once again, the subtraction of the log-transformed images provides
the MP spatial distribution. In an attempt to simplify the procedure and analysis as much as possi-
ble, a number of investigators have chosen to rely on a single image captured at, or close to, the peak
wavelength in the MP absorption spectrum (Schweitzer et al. 2002). Such images always display a
decreased intensity in the foveal part of the image and it is tempting to attribute this entirely to the
MP. However, images captured in the green part of the spectrum prior to bleaching usually show a
decreased foveal intensity, due to the absorption by cone photopigments, which peaks exactly where
MP peaks.
A recent attempt to overcome this problem and still retain a relatively straightforward procedure
has been reported by Bone et al. (2007a). Using a standard digital retinal camera in conjunction
with multi-band-pass i lters, it was possible to extract images of the retina at four different wave-
lengths from just two captured images. The retina was modeled as a sequence of four spatially
varying, absorbing layers backed by a spectrally neutral rel ector, the sclera. The layers consisted
of MP, cone photopigments, rod photopigment, and melanin. In accordance with the model, and
using published extinction spectra of the absorbing pigments, the four monochromatic images were
transformed logarithmically and then combined linearly to yield optical density distribution maps
of not only MP, but also cone and rod photopigments, and melanin. Because of the susceptibility of
retinal cameras to intraocular light scatter that results in less than perfect images, the method may
be unsuitable for older subjects for whom light scatter is more pronounced.
5.3.2 L
IPOFUSCIN
A
UTOFLUORESCENCE
-B
ASED
M
ETHOD
Posterior to the neural retina, and therefore to the MP, is the retinal pigmented epithelium (RPE).
Lipofuscin is a l uorescent material that is sequestered in RPE cells. The l uorescence can be excited
by wavelengths in the ~400-570 nm range, which includes the range of MP absorbance, and emis-
sion is in the ~520-800 nm range, which excludes MP absorbance. The principle behind the autol u-
orescence-based method of measuring MP is that an exciting wavelength close to the MP absorption
maximum will be attenuated by the MP resulting in an MP-dependent intensity of l uorescence
emission (Delori et al. 2001).
The methods can be subdivided into the one- and two-wavelength methods. In an application of
the one-wavelength method, an SLO is modii ed so that l uorescence images of the retina can be
obtained using the 488 nm line of an argon laser as the exciting wavelength (Robson et al. 2003,
Trieschmann et al. 2003). A barrier i lter that transmits only wavelengths above 560 nm is used
in conjunction with the detector so as to exclude the excitation light. The resulting images show a
decreased intensity in the foveal region due to absorption of the exciting light by the MP and, con-
sequently, a decrease in l uorescence emission. By comparing the intensity of l uorescence at any
point in this region with the intensity at, say, 6° eccentricity where MP density is assumed to be neg-
ligible, a two-dimensional MP optical density distribution at 488 nm can be generated. If needed,
the distribution can be multiplied by the ratio of MP extinction coefi cients,
K
460
/
K
488
, to obtain the
MP optical density distribution at the peak wavelength, 460 nm.
An assumption inherent in the one-wavelength method is that the distribution of lipofuscin is
uniform throughout the area of the retina being analyzed. Certainly if lipofuscin concentration
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