Raman Microscopy and Imaging: Applications to Skin Pharmacology and Wound Healing - Emerging Raman Applications and Techniques in Biomedical and Pharmaceutical Fields - page 375

Fig. 15.11. a Factor loadings constructed from Raman spectra specific to the gen-
eral area of the wound. The amide I and III contours of factors 1 and 2 are charac-
teristic of high collagen and high keratin levels, respectively. b Factor score images
reveal that collagen (factor 1) in the wounded area is pressed up from the dermis,
while the keratin (factor 2) in the non-wounded area arises from the SC and from
the viable epidermis

from both the SC and from the viable epidermis. The spatial distribution
of components in the wounded area of skin is best investigated with factor
analysis conducted over the 700-1010 cm − 1 region. Factor loadings specific to
the wounded area are shown in Fig. 15.12a and corresponding score images
are presented in Fig. 15.12c. In the loading plots (Fig. 15.12a), the doublet
of doublets between 830 and 960 cm − 1 in all three traces is characteristic of
collagen; these bands have been assigned to vibrations of Pro and Hyp amino
acids and the C-C stretch of the protein backbone [44]. Factors 2 and 3,
whose loadings are quite similar throughout the spectral range, have score
plots (Fig. 15.12c, middle and bottom) in the wounded area that generally
complement each other spatially. The molecular structure differences produc-
ing the slightly altered loadings are currently under investigation, and may
be related to altered collagen hydration levels. Factor 1 in Fig. 15.12a is dis-
tinguished from the others by the presence of the band at 906 cm − 1 and a
relatively stronger Phe band at 1004 cm − 1 . To explore the origin of this fac-
tor, the average spectrum of pixels having low scores in the area surrounding
the higher scoring regions (in Fig. 15.12c, top image) was subtracted from the
average spectrum of pixels with high scores for f1. The resulting difference
spectrum (shown in Fig. 15.12b) reveals that f1 contains the elastin compo-
nent in the wound bed. The spectrum is essentially identical to a published
spectrum of bovine elastin [44, 45]. It is likely that the elastin component was
not identified as a separate factor due to the small diameter of the elastin
fiber bundles (

m) in the dermis compared to the spot size of the
excitation laser. Thus, factor 1 (Fig. 15.12a) contains features of both elastin
and collagen.
Overall, confocal Raman microscopy demonstrates the ability to track
changes in the spatial distribution of two major skin proteins (collagen and

∼ 0 . 25-1 . 0 μ