Raman Spectroscopy of Ocular Tissue - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 304

studies involving subjects with lens implants. Therefore, the Raman method
would be well suited, for example, in important nutritional supplementation
trials, studies in which significant increases in individual MP levels have been
demonstrated to be achievable in a time span of about 12 months [45].
While the resonance Raman methods are rapid, non-invasive, and highly
specific for the non-invasive measurements of MP, the Raman response is still
relatively weak. To compensate for this, dilated pupils with 6 mm diameter or
larger have to be used. Although common practice in eye examination, this
requirement is inconvenient for rapid measurements of large populations. This
drawback can be avoided in the recently developed, non-mydriatic version of
the lipofuscin fluorescence imaging (autofluorescence imaging) method [46],
and potentially also in reflection-based imaging methods which are currently
under development [47]. Both methods benefit from the lower excitation light
levels required in fluorescence and reflectance detection schemes.
Autofluorescence imaging, AFI, detects the light emitted from lipofuscin
in the retinal pigment epithelium and derives the concentration of MP only
indirectly via fluorescence excitation spectroscopy [48-50]. The method takes
advantage of the fact that both lipofuscin and MP have overlapping absorp-
tion bands in the blue wavelength region, and that light excitation with blue
wavelengths leads to a strong lipofuscin emission band in the red/near-IR
wavelength region. Furthermore, it uses the fact that lipofuscin is relatively
evenly distributed throughout the retina, while MP is concentrated only in
the macula.
Using blue light illumination of the retina with a large-diameter disk cen-
tered on the macula and extending to peripheral regions, the AFI method
compares the lipofuscin fluorescence intensities outside and inside the macula.
Outside the macula, the fluorescence is not attenuated, since the excitation
light can stimulate the lipofuscin fluorescence without any attenuation. Inside
the macular region, however, MP located in the anterior layers can attenuate
the excitation light. This results in a corresponding reduced lipofuscin fluo-
rescence response. MP optical density levels can then be determined from the
logarithm of the ratio of lipofuscin fluorescence levels inside and outside the
macula.
AFI has an additional advantage over the Raman approach since the pe-
ripheral reference location allows one to eliminate, in first order, any po-
tentially confounding attenuation effects arising from anterior optical media.
Also, AFI-derived MP results can be obtained with one image only. On the
other hand, this method is not as specific, however, compared to Raman, and
care has to be taken to avoid potentially confounding fluorescence effects from
anterior ocular media such as the lens.
In Fig. 12.16 the main results are compared for MP distributions and
concentrations obtained with both the RRI and AFI methods for an identical
subgroup of subjects. Compared to the RRI image, the AFI image is nearly
identical, with the exception of a smoother appearance of the distribution. For
a group of 16 subjects, MP levels obtained with both methods were spatially