Raman Spectroscopy of Ocular Tissue - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 283

The frequency of the amide I peak observed in the lens is sensitive to
protein secondary structure. From its absolute position at 1672 cm − 1 ,whichis
indicative for an antiparallel pleated β -sheet structure, and the absence of lines

in the 1630-1654 cm − 1 region, which would be indicative of parallel β -sheet

and -helix structures, the authors could conclude that the lens proteins are all

organized in an antiparallel, pleated

α

-sheet structure [3]. Schachar and Solin
[4] reached the same conclusion for the protein structure by measuring the
amide I band depolarization ratios of lens crystallins in excised bovine lenses.
Later, the Raman-deduced protein structure findings of these two groups were
confirmed by x-ray crystallography.
Further results from these Raman studies showed that the lens nucleus
and cortex had somewhat different amino acid compositions. It could be con-
cluded that the nuclear portion has highest concentration of

β

γ -crystallin, and

that the content of -crystallin increases significantly from the nucleus to the

cortex. Furthermore, it was found that sulfhydryl groups and

α

-conformation
are unaffected in the conversion from a transparent to totally opaque lens
by heat denaturation. This indicates that the opacification of the lens does
not necessarily involve the oxidation of sulfhydryl groups or conformational
changes [3].
Using laser excitation with visible wavelengths, cataracteous lens regions
are typically generating large, overwhelming fluorescence contributions to the
Raman spectra due to fluorescing protein aggregates with high molecular
weight. This limits Raman experiments usually to the cortical lens regions,
where the cataracts and corresponding fluorescence intensities are diminished
relative to the nuclear region of the lens. The fluorescence can be reduced
to some degree by choosing laser excitation in the red wavelength region [6].
While it was possible to extend lens Raman studies to the living rabbit eye
[7], use with living human eye lenses has not yet been possible due to the
required high light levels (

β

∼ 15 mW at cornea) and rather long exposure times

( 2 min).

Duindam et al. [8] investigated sharply localized, small opaque areas in the
human lens, termed as focal opacities, which are progressing slowly over a life-
time, and which may be precursors to mature cataracts. Working with human
donor eyes, the authors were able to detect changes in proteins, phospholipids,
and cholesterol in these opacities, employing a combination of high-resolution
techniques, including Raman microspectroscopy, cytochemistry, and trans-
mission electron microscopy. With electron microscopy they could identify
normal and opaque lens regions, and then compare these areas with their
Raman responses. The difference ∗ Raman spectrum, obtained from spectra
within the opacity and from spectra of normal tissue areas, is reproduced in
Fig. 12.3. Compared with normal tissue regions, it shows that opacities con-
tain unchanged protein content, but significantly increased aliphatic CH and
CH 2 chains and reduced aromatic amino acid content. Also, the opacities con-
tained elevated levels of cholesterol, and there was evidence for the formation
of disulfide (S-S) bridges [8].

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