Chemical Structural Analysis of Bone by Spectroscopy - Vibrational Spectroscopy for Tissue Analysis - page 190

to processes that may alter structure and/or composition. The ideal situation
would be one in which minimal tissue preparation is required and physiologic
conditions are maintained as closely as possible, and this can be achieved by
employing both the Raman and infrared spectroscopic techniques [10].
Raman Spectroscopy of Bone
Raman spectroscopy is increasingly becoming recognized as a significant ana-
lytical method for biomedical applications and has the following advantages:
1. The need for tissue preparation is minimal. The measurement itself
is nondestructive to the tissue and only very small amounts of mate-
rial (micrograms to nanograms) are required.
2. “Wet” samples at atmospheric pressure can also be analysed, which
is representative of a physiologic condition.
3. Molecular (ultrastructural-level) information is available from both
infrared and Raman techniques, allowing investigation of functional
groups, bonding types, and molecular conformations. The use of
interferometers and Fourier transformation has allowed the realiza-
tion of several spectrochemical advantages over conventional, dis-
persive spectrophotometers, such as increased signal throughput,
greater frequency precision, and a decrease in the time required to
obtain a spectrum [11].
However, by the incorporation of the Nd:YVO 4 near-infrared laser into
Raman instrument design, the most significant feature of such technology
is the virtual elimination of fluorescence from most biological samples. The
problem of fluorescence previously restricted the Raman spectrochemical
analysis of bone, and it was suggested that the fluorescing species lie within
the organic phase of the tissue [12]. The fluorescence effect obscured much of
the Raman signal originating from the analyte and restricted the applicability
of the technique.
The problem of fluorescence was overcome previously by the deproteination
of bone samples to remove the organic phase. Walters et al. [12] obtained
conventional Raman and FTIR spectra of cortical bone from an ox femur.
A conventional Raman investigation was also performed by Gevorkyan et al.
[13] on the cortical bone of the human femur and on hydroxyapatite samples.
The values quoted for the various phosphate peaks were in broad agree-
ment with those reported by others [6,14-16], and the values given for the
corresponding peaks arising from the bone tissue agreed well with those of
the hydroxyapatites.
The two studies of bone tissue described earlier [12,13], although revealing
some detail concerning the mineral phase of the tissue, were affected by