Raman Spectroscopy of Ocular Tissue - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 285

short-range liquid-like order of the crystallin proteins. As a measure for pro-
tein content, they used the ratio between the Raman intensity of the aliphatic
CH stretch mode of proteins at 2935 cm − 1 and the water OH stretch mode
at 3390 cm − 1 [11]. The authors were able to establish that changes in the
amount of light scattering along the visual axis of the lens are not explainable
with protein concentration effects. Rather, these effects need to be attributed
to local changes in protein conformation, such as polydispersity, aggregation,
and non-sphericity of the crystallins. These changes vary among and within
human lenses and change with age, leading to significant age-related and local
variations in light scattering.
The prospects of influencing the structural changes of lens protein with
vitamin E effects were investigated by Shih et al. [12] in lenses of tilapia.
Using FT-Raman spectroscopy they explored potential structural changes of
lens proteins occurring as an effect of dietary vitamin E supplementation.
The authors found that while the protein secondary structure remained unal-
tered, significant changes occurred in the microenvironment of the lens con-
stituents tyrosine, tryptophan, and thiol compounds. The changes were more
pronounced in the cortex than the nucleus. The authors detected a close con-
nection between vitamin E intake and a Raman band at 1090 cm − 1 , attributed
to the lens membrane. Potentially, the strength of this band could be used to
investigate the role of vitamin E in the protection of lens membranes against
external stresses.

12.3 Hydration Studies of the Cornea

The cornea accounts for about 70% of the refractive power of the un-
accommodated human eye, and therefore plays an important role in maintain-
ing optimal vision. Apart from water, the cornea consists of

15% collagen
proteins, which are mostly comprised of glycine, an aliphatic amino acid, and
proline, the only cyclic amino acid. Areas where Raman spectroscopy can con-
tribute include investigations of collagen proteins, corneal hydration status,
and the pharmacokinetics of topically applied ocular hydration drugs.
High-quality Raman spectra of corneal collagen proteins with conventional
Raman spectroscopy had been dicult to obtain due to strong interferences
from interior autofluorescence responses, and due to excessively high laser
excitation levels. However, some progress in this area occurred after the de-
velopment of Fourier-based IR and Raman spectroscopy techniques. These
techniques avoid the influence of fluorescence on the spectra and also feature
higher sensitivity. As a result, good-quality Raman spectra could be obtained
from excised animal corneas, as shown in Fig. 12.4 as an example for a rabbit
cornea [13]. Comparing the animal Raman spectra with those of pure type
I collagen and other connective tissue components, such as proteoglycans,
hyaluronic acids, chondroitin sulfates, and keratin sulfates, it was possible
to assign the observed amide I and III responses of the animal spectra to

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