1. Clustering of infrared spectra reveals histological zones in intact articular cartilage 
Yevgeniya Kobrina, Lassi Rieppo, Simo Saarakkala, Jukka S. Jurvelin, Hanna Isaksson 
Osteoarthritis and Cartilage 
Volume 20, Issue 5, Pages (May 2012)
DOI: /j.joca
Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
(A) A schematic representation of the articular cartilage structure (modified from43). The superficial zone (SZ), middle zone (MZ), deep zone (DZ) and calcified zone (CZ) of the tissue are indicated; (B) A typical FTIR microspectroscopy spectrum of rabbit and bovine articular cartilage. Spectral peaks of interest are indicated (amide I, amide II, amide III and proteoglycan). Spectra are scaled so that the maximum value of the amide I peak (1585–1720 cm-1) equals one; (C) Pre-processing steps. Bone spectra and outliers were removed from the images using principal component analysis (PCA). The first, most discriminative, PCA image was built based on the proteoglycan (968–1140 cm−1) region of infrared spectra and clustered using the fuzzy c-means algorithm. Pixels assigned to bone and outliers were removed. Integrated collagen absorbance images show the sample before and after preprocessing.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Fuzzy C-means clustering results for one rabbit sample based on the (A) amide and (B) proteoglycan (PG) spectral regions. The integrated absorbance images, corresponding false-color clustering maps and mean spectra for each cluster are shown for each spectral region. The toluidine blue stained histological image is shown as a reference.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

4. Fig. 3
Fuzzy C-means clustering results for one bovine sample based on the (A) amide and (B) proteoglycan (PG) spectral regions. The integrated absorbance images, corresponding false-color clustering maps and mean spectra for each cluster are shown for each spectral region. The safranin O stained histological image is shown as a reference.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Fuzzy clustering maps of a (A) rabbit and (B) bovine samples, obtained using the amide spectral region. Degrees of membership for each pixel to each of the three clusters are given, and gradual transition between clusters is revealed. The “hard” clustering map was calculated based on fuzzy map, so each pixel is assigned to the cluster with the maximum membership value.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

6. Fig. 5
Mean normalize raw spectra of clusters of (A) rabbit and (B) bovine samples. Magnified images show shape differences in spectra on AI and AII regions. Spectral differences between clusters are enhanced by the corresponding second derivative spectra for (C) rabbit and (D) bovine samples.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
(A) Average integrated peak areas of AI, AII, proteoglycan (PG) and (B) average PG/AI and AI/AII ratios, for each of the three clusters. The data is shown as medians with 95% confidence interval of from all the seven rabbit AC samples. Significant differences based on the Mann–Whitney U test are indicated.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions

8. Fig. 7
(A, B) Histological sections and corresponding (C, D) parallelism index of collagen fibers calculated from polarized light microscopy (PLM) images for one rabbit and one bovine samples6,28. The arrow indicates an extra lamina in the bovine sample. Parallelism index6 of 1 corresponds to completely parallel and 0 corresponds to completely random orientation of fibers inside one particular pixel. (E) The magnified image displays the histological zones of articular cartilage determined by PLM. (F, G) FCM maps of the rabbit and bovine samples show good correspondence between clustering and PLM images.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2012 Osteoarthritis Research Society International Terms and Conditions