6.2.4  Raman spectroscopy

Elastic scattering of light, i.e., the scattered ray has the same wavelength as the incoming ray, is very common.

6  However, in some cases, in addition a small portion of the incident light is scattered inelastically, i.e., at longer or shorter wavelengths. This process is known as Raman scattering [154]. The light frequency shifts correspond to the vibrational energies of the molecules at which the light scatters. It can be used to obtain specific optical fingerprint spectrum of molecules. In general, Raman spectroscopy in biological systems yields quite complex spectra because of the wide variety of chemical compounds present. Moreover, these spectral peaks tend to be of weak intensity, because only a small proportion of the scattered light is Raman shifted. However, under certain situations when the incident light overlaps with a molecule’s major absorption band, if the molecule is Raman active, and if there is no interfering fluorescence, the Raman signals can be resonantly enhanced by many orders of magnitude. In particular, carotenoids have intense absorption bands in the blue/green wavelength range that result in large resonant enhancements [124]. Fortunately, no other biological molecules found in significant concentrations in human ocular tissues exhibit similar resonant enhancement with blue laser excitation. This makes the in vivo carotenoid Raman spectra remarkably free from confounding responses. Bernstein et al. have shown that this technique can be used to measure the MP in vivo in the human eye [155–157]. Although there were some concerns about this technique [127–130, 158, 159], it seems to give reliable results that compare well with HFP, provided a proper protocol is used [131].

6.2.5  How do different techniques compare

All techniques have been shown to correlate well. However, correlation analyses only reveal the strength of relation between techniques but they do not necessarily reveal agreement in an absolute sense. In fact, it is unlikely that different methods will agree quantitatively, since they use different principles and probe different retinal areas. Indeed, systematic, but explainable differences between different methods are observed [71, 102, 111, 131, 134, 160–163]. For instance, optical technique probes a retinal area and provides MPOD estimates averaged over this particular area. Further, because of its spatial peakedness, the size of the retinal field probed has a major effect. This contrasts with HFP, in which subjects make their judgment as to the absence/presence of flicker at the edge of the flickering target. As a result in HFP, MPOD will be biased toward lower values [110, 111, 164–166].

Summary for the Clinician

■There are psychophysical and optical techniques to determine the MPOD, which give reliable results.

■The most widespread are the psychophysical techniques, where the subject adjusts color or luminosity, generally through a minimum flicker task. The advantage of these approaches is that there is no need for pupil dilation. A disadvantage is that they are rather time consuming, and the task, in particular, when making a match in the peripheral retina, is not trivial.

■There are several optical approaches to obtain the MPOD. They all rely on the analysis of light returning from the retina, either on spectral analysis, autofluorescence (AF), or on Raman spectroscopy. The advantage of these techniques is their more objective approach and measuring time. A disadvantage for some setups (but not all) is that they require pupil dilation. Moreover, not all are commercially available.

■All the techniques have been shown to correlate well. However, they do not reveal agreement in an absolute sense. Since different methods probe the MP differently, systematic, but explainable differences are observed. Therefore, in longitudinal studies an individual should stick to one and the same method.

■For all methods, a proper setup and protocol should be used to avoid measurement errors like the use of improper frequencies in heterochromatic flicker photometry, or the influence of stray light in reflectance spectroscopy or the influence of pupil width in resonant Raman spectroscopy.

6.3  Imaging

All techniques described earlier started off to have MPOD estimates over a well-defined retinal area. In most of the cases, this has been 1° field. To measure the spatial extent of the MP, setups have been developed for imaging a wider area and with a finer spatial resolution.

6.3.1  Heterochromatic Flickerphotometry

Using HFP it is time-consuming to measure the full spatial distribution of the MP. The reason for this is twofold. First, the stimulus, i.e., the target that the subjects have to reduce to a null point, needs a finite size. Second, as already

mentioned, the method is rather time-consuming (about 30 min for 6 eccentricities [167]). This may be a limiting factor for both large epidemiological trials and a clinical setting. Nevertheless, some studies have been presented with a low spatial solution. Hammond et al. were the first to study the spatial profile by HFP in normal subjects at 0, 0.5, 1, 2, 3, and 4° and a reference point at 5.5° [66]. Stringham et al. measured patients with intermediate stages of agerelated macular degeneration (AMD) [167]. Patients with visual acuity as poor as 20/80 were included. MPOD was measured at 15¢, 30¢, 1º, 1.5º, 3º, and 5º along the horizontal meridian and a reference location at 7º eccentricity (see Fig. 6.4). Spatial profiles of MPOD were similar to those that have been measured with HFP in subjects without retinal disease. Nolan et al. measured MPOD at eccentricities of 0.25, 0.5, 1, 1.75, 3, and 5° along the horizontal meridian relative to a reference location at 7° eccentricity and looked for correlations with foveal architecture (see above) [76].

ratio in AF maps, in general, several AF images are averaged, whereas in reflectance image a single image suffices. However, image registration algorithms are well developed and images can be aligned easily. Using this approach, a high spatial resolution can be achieved and many studies have been published, both in normal subjects and in patients with different diseases of the retina.[7, 67, 70, 71, 73, 81, 108, 146]. Figure 6.10 shows an example of autofluorescence maps at 488 and 514 nm and the resulting MPOD map. The AF image at 488 nm is substantially brighter than the one at 514 nm. The AF method uses the intrinsic fluorescence of the lipofuscin with its absorption peak at 490 nm. This makes the 488 nm more efficient than the 514 nm excitation. Further, in this particular setup, the filters to capture the AF had a cut-off wavelength of 510 nm for the blue excitation and a cut-off wavelength for the green excitation. As a result, more fluorescence was captured from

6.3.2  Fundus Reflectance

The technique of comparing the reflection at the macular region and at a peripheral region can be exploited to obtain MPOD maps. The most common method is the use of a Scanning Laser Ophthalmoscope (SLO) with 488 and 514 nm Argon laser wavelengths to obtain fundus reflectance maps [67, 81, 95, 121, 146, 168, 169]. Since the lens and the macular pigment are the only absorbers in this wavelength region, digital subtraction at these two wavelengths of log reflectance can provide density maps of the sum of both absorbers. MPOD is assumed to be negligible at a peripheral site. If this site is used to provide an estimate for the lens density, the mean MPOD at the fovea can be calculated [95, 168]. Figure 6.10 shows an example of these maps and the resulting MPOD map. Using this technique, the MPOD spatial can be imaged easily and with a high resolution. It allowed the quantification of a ring-like structure on top of the decreasing function with eccentricity as discussed earlier (see Fig. 6.5) [67]. A confocal setup, like the SLO is necessary to obtain reliable results. Attempts to use a fundus camera yielded rather low MPOD values, probably due to stray light [118, 145].

6.3.3  Autofluorescence

The most widespread method to obtain a detailed MPOD is using AF, probably because of the availability of commercial devices to do so. Like in fundus reflectance, here AF maps are made at different wavelengths, and a MPOD map is created by digital subtraction of the two log AF maps. Because of the low signal-to-noise

Fig. 6.10  Macular pigment optical density (MPOD) as a function of eccentricity from reflectance (black) and autofluorescence (red) maps. MPOD maps obtained from reflectance (left) and autofluorescence (right) are shown as insets. The MPOD distribution was assumed to be circularly symmetric and MPOD was determined as a function of eccentricity by calculating for each pixel the distance to the peak. Solid lines: results of a model fits. Reprinted with permission of Investigative Ophthalmology and Visual Science

the 488 nm excitation than from the 514 excitation. Finally, the voltage of the photomultiplier tube may differ between the two wavelengths. All these phenomena may result in an apparent difference in gain between

6  the 488 and 514 nm maps. However, MPOD maps are obtained by digital subtraction at two wavelengths of log AF, which implies only an offset in the MPOD map. Analyzing the MPOD maps, it is assumed that the MPOD essentially vanishes at 8°; thus, at this point the optical density is defined to be zero. Figure 6.5 shows MPOD as a function of eccentricity obtained from AF maps by Berendschot et al. [67] showing the same ringlike structure as in those obtained from reflectance maps. Similar MPOD maps from AF images were obtained independently by Delori et al. [180] and WolfSchnurrbusch et al. [181].

Some studies only measured AF maps at 488 nm, without a reference measurement at a wavelength that is not absorbed by the MP [70, 174, 182,]. Here, the observed MPOD map may be diluted by varying lipofuscin and melanin concentrations as a function of eccentricity, the exact shape of which is unknown. It makes these results difficult to interpret [183].

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b

6.3.4  Raman spectroscopy

Resonant Raman spectroscopy has also become available in imaging mode [125], even without the need of pupil dilation [184]. Using laser excitation of lutein and zeaxanthin at 488 nm, and sequential camera detection of light emitted back from the retina at the strongest Raman peak position and at an off-peak position, resonant Raman maps of MP are determined (See Fig. 6.11). The resolution of these maps allows the determination of fine structures like the rings seen by fundus reflectance and AF maps (see Fig. 6.12) [184].

Summary for the Clinician

■Both psychophysical and optical techniques can be used to map the spatial distribution of the MP. Psychophysical techniques are rather timeconsuming in measuring the MPOD spatial profile, and still have a rather low resolution (about 30 min for 6 eccentricities only). Optical techniques are more suited since they can produce MPOD maps with a high resolution within minutes.

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2000

Distance (µm)