Application of second derivative spectroscopy for increasing molecular specificity of Fourier transform infrared spectroscopic imaging of articular cartilage.
OBJECTIVE
Fourier transform infrared (FT-IR) spectroscopic imaging is a promising method that enables the analysis of spatial distribution of biochemical components within histological sections. However, analysis of FT-IR spectroscopic data is complicated since absorption peaks often overlap with each other. Second derivative spectroscopy is a technique which enhances the separation of overlapping peaks. The objective of this study was to evaluate the specificity of the second derivative peaks for the main tissue components of articular cartilage (AC), i.e., collagen and proteoglycans (PGs).


METHODS
Histological bovine AC sections were measured before and after enzymatic removal of PGs. Both formalin-fixed sections (n = 10) and cryosections (n = 6) were investigated. Relative changes in the second derivative peak heights caused by the removal of PGs were calculated for both sample groups.


RESULTS
The results showed that numerous peaks, e.g., peaks located at 1202 cm(-1) and 1336 cm(-1), altered less than 5% in the experiment. These peaks were assumed to be specific for collagen. In contrast, two peaks located at 1064 cm(-1) and 1376 cm(-1) were seen to alter notably, approximately 50% or more. These peaks were regarded to be specific for PGs. The changes were greater in cryosections than formalin-fixed sections.


CONCLUSIONS
The results of this study suggest that the second derivative spectroscopy offers a practical and more specific method than routinely used absorption spectrum analysis methods to obtain compositional information on AC with FT-IR spectroscopic imaging.
Department of Applied Physics, University of Eastern Finland, Kuopio, Finland; Department of Clinical Neurophysiology, Kuopio University Hospital, Kuopio, Finland. Electronic address: lassi.rieppo@uef.fi.
L
L
Rieppo
Department of Diagnostic Radiology, Institute of Diagnostics, University of Oulu, Oulu, Finland; Department of Medical Technology, Institute of Biomedicine, University of Oulu, Oulu, Finland. Electronic address: simo.saarakkala@oulu.fi.
S
S
Saarakkala
Institute of Biomedicine, Department of Anatomy, University of Eastern Finland, Kuopio, Finland. Electronic address: tommi.narhi@fimnet.fi.
T
T
Närhi
Institute of Biomedicine, Department of Anatomy, University of Eastern Finland, Kuopio, Finland. Electronic address: heikki.helminen@uef.fi.
H J
HJ
Helminen
Department of Applied Physics, University of Eastern Finland, Kuopio, Finland. Electronic address: jukka.jurvelin@uef.fi.
J S
JS
Jurvelin
Institute of Biomedicine, Department of Anatomy, University of Eastern Finland, Kuopio, Finland; Iisalmi Hospital, Iisalmi, Finland. Electronic address: jarno.rieppo@uef.fi.
J
J
Rieppo
D000818:Animals; D002358:Cartilage, Articular / Q000033:anatomy & histology / Q000378:metabolism*; D002417:Cattle; D024043:Collagen Type II / Q000378:metabolism; D015925:Cryopreservation; D005404:Fixatives; D005557:Formaldehyde; D008297:Male; D011509:Proteoglycans / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D016707:Tissue Fixation
D024043:Collagen Type II; D005404:Fixatives; D011509:Proteoglycans; D005557:Formaldehyde