Figure 2shows a cluster plot for the principal spectral components for the SC, TA cell, and TD cell populations. It is clear that the three different cell types of interest formed discrete clusters. The cluster for TD cells was the most densely aggregated and was also distinctly spatially separated from both SCs and TA cells. Although SC and TA cell populations also formed distinct clusters, there was some slight overlap (≈2%) between these two populations.
Figure 3Ashows that the median spectra for the SC and TA cell populations exhibited differences, especially in the 1490 to 1000 cm
−1 region of the spectrum that chiefly relates to changes in nucleic acid conformation.
14 Figure 3Bis a difference spectrum (i.e., the difference between the median spectra of the SC and TA specimens), which enabled the identification of those regions that exhibited the greatest difference between the cell types. Regions that showed the greatest spectral differences (>0.05 absorption units) were subjected to the Mann-Whitney test, and spectral peaks with a significance level of
P < 0.001 are indicated in
Figure 3B . The positions of these peaks correlated with the differences in the particular molecular groups. Those peaks identified as significant were the peak at 1714 cm
−1, which is associated with C⋕O stretching vibrations of nucleic acids
16 ; the peak at ≈1600 cm
−1, which is at the edge of the spectral region sensitive to changes in nucleic acid base pairing
18 ; the peaks at ≈1450 to 1480 cm
−1, which are due to CH
2 scissoring and CH
3 asymmetric bending vibrations of lipids, proteins, and nucleic acids
16 ; the peaks at ≈1380 cm
−1 and 1260 cm
−1, which occur in a region linked to cell cycle changes
14 15 ; and finally the peak at ≈1225 cm
−1, which has been linked to PO
2 − asymmetric stretching vibrations of nucleic acids.
16
Figure 4Ashows that the median spectrum for TA cells compared with that derived from TD cells also exhibit apparent differences.
Figure 4Bis a difference spectrum, which enabled the identification of those regions that exhibited the greatest differences between the cell types. Regions which showed the greatest spectral differences (>0.05 absorption units) were subjected to the Mann-Whitney test and spectral peaks with a significance level of
P < 0.001 are highlighted in
Figure 4 . The positions of these peaks are correlated with differences in particular molecular groups. Those peaks identified as significant are listed as follows: the amide I peak at ≈1650 cm
−1, which is associated with proteins and changes in protein secondary structure (α-helical/β-pleated)
16 ; the peak in the ≈1120 cm
−1 region, which is associated with RNA expression
14 ; the peak centered around 1080 cm
−1 which has been linked to absorption by protein and RNA
17 ; and the peak around 1030 cm
−1, which is associated with glycogen content.
13 14