Quantitative Methods for the Determination of Carotenoids in the Retina - Carotenoids: Physical, Chemical, and Biological Functions and Properties - page 83

peaked at the fovea, it would lead to enhanced l uorescence and an underestimate of the central
MP optical density. To eliminate this problem, Delori et al. (2001) introduced the two-wavelength
method. In this method, two exciting wavelengths provided by the argon laser can conveniently
be used (Trieschmann et al. 2006). One wavelength (e.g., 488 nm) is attenuated by the MP; the
other (514 nm) is minimally absorbed. Using the longer wavelength, the distribution of l uorescence
reveals any nonuniformity in the concentration of lipofuscin and is unaffected by MP. Multiplying
this distribution by the ratio of l uorescence efi ciencies of lipofuscin, F 460 / F 514 , at the two wave-
lengths, we obtain the distribution of l uorescence due to excitation by the shorter wavelength as
it would appear in the absence of MP. Comparing this with the actual distribution of l uorescence
obtained with the shorter exciting wavelength allows one to compute the optical density of the MP.
In fact, what is reported is the difference in optical density between any point in the retina and a
parafoveal reference point where MP optical density is assumed to be negligible. In this calcula-
tion, the ratio of l uorescence efi ciencies of lipofuscin, F 460 / F 514 , assumed to be the same at the
two retinal locations, is eliminated from the i nal expression. However, the l uorescence of lipofus-
cin is due to the presence of more than one l uorophore (Parish et al. 1998) and, if the composition
of lipofuscin changes with retinal location, it is possible that the ratio of l uorescence efi ciencies
is not constant across the retina. In this case, an error would occur in the calculated MP optical
density.
5.3.3 R ESONANCE R AMAN S PECTROSCOPY
When carotenoids such as lutein and zeaxanthin are excited by wavelengths in the ~450-550 nm
range, they exhibit particularly strong resonance Raman signals that can be used to quantify the
amount of carotenoid present. The application of this technique for quantifying the macular carote-
noids has been developed, thereby providing another noninvasive physical method for MP measure-
ment. A detailed description of this method is given in Chapter 6.
ACKNOWLEDGMENTS
Support provided by NIH grants S06 GM08205 and R25 GM61347.
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