Application of second derivative spectroscopy for increasing molecular specificity of fourier transform infrared spectroscopic imaging of articular cartilage  L. Rieppo, S. Saarakkala, T. Närhi, H.J. Helminen, J.S. Jurvelin, J. Rieppo  Osteoarthritis and Cartilage  Volume 20, Issue 5, Pages 451-459 (May 2012) DOI: 10.1016/j.joca.2012.01.010 Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Infrared absorption spectra from AC (solid line) and type II collagen (dashed line) (A). Pure compound spectrum of chondroitin sulphate (solid line) and a difference spectrum (see Data analysis in Materials and Methods) computed by subtracting the mean spectrum after the enzyme treatment from the mean spectrum before enzyme treatment of formalin-fixed samples (dashed line) (B). Osteoarthritis and Cartilage 2012 20, 451-459DOI: (10.1016/j.joca.2012.01.010) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 The mean second derivative spectra of AC of formalin-fixed sections (A). The mean relative change (± standard deviation) in the amplitude of second derivative absorption peaks in formalin-fixed sections (black squares) (B). For comparison, the mean relative changes in cryosections for the two peaks that changed the most are marked with white squares. Osteoarthritis and Cartilage 2012 20, 451-459DOI: (10.1016/j.joca.2012.01.010) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 The changes in the total absorbance, amide I absorbance, carbohydrate region, the ratio of carbohydrate region to amide I as well as the changes in collagen- and PG-specific second derivative peaks. White stars indicate a statistically significant difference in the amplitude caused by removal of PGs (Wilcoxon signed-rank test, P < 0.05). Black stars indicate a statistically significant difference between formalin-fixed sections and cryosections (Mann–Whitney U-test, P < 0.05). Two stars indicate statistical significance level of P < 0.01. Osteoarthritis and Cartilage 2012 20, 451-459DOI: (10.1016/j.joca.2012.01.010) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Depth-wise distributions of the heights of two PG-related and two collagen-related second derivative peaks. The distributions before the removal of PGs are marked with squares and solid lines, whereas the distributions after the removal of PGs are marked with triangles and dashed lines. Profiles are group means of formalin-fixed samples (n = 10). Standard deviations are shown for five points for each distribution. (A) The height of the PG-related second derivative peak at 1376 cm−1. (B) The height of the PG-related second derivative peak at 1064 cm−1. (C) The height of the collagen-related second derivative peak at 1202 cm−1. (D) The height of the collagen-related second derivative peak at 1336 cm−1. Osteoarthritis and Cartilage 2012 20, 451-459DOI: (10.1016/j.joca.2012.01.010) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Safranin O-stained section before the enzyme treatment (A), safranin O-stained section after the enzyme treatment (B). Chemical FT-IR spectroscopic imaging maps of untreated AC (C–F): the height of the second derivative peak at 1064 cm−1 (sulphated PGs) (C), the height of the second derivative peak at 1376 cm−1 (all PGs) (D), the height of the second derivative peak at 1202 cm−1 (collagen) (E), the height of the second derivative peak at 1336 cm−1 (collagen) (F). Osteoarthritis and Cartilage 2012 20, 451-459DOI: (10.1016/j.joca.2012.01.010) Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions