Noninvasive Raman spectroscopy of rat tibiae: approach to in vivo assessment of bone quality

Abstract

We report on in vivo noninvasive Raman spectroscopy of rat tibiae using robust fiber-optic Raman probes and holders designed for transcutaneous Raman measurements in small animals. The configuration allows placement of multiple fibers around a rat leg, maintaining contact with the skin. Bone Raman data are presented for three regions of the rat tibia diaphysis with different thicknesses of overlying soft tissue. The ability to perform in vivo noninvasive Raman measurement and evaluation of subtle changes in bone composition is demonstrated with rat leg phantoms in which the tibia has carbonated hydroxylapatite, with different carbonate contents. Our data provide proof of the principle that small changes in bone composition can be monitored through soft tissue at anatomical sites of interest in biomedical studies.

Fig. 1

Noninvasive Raman spectroscopy of a rat tibia. (a) The probe holder is fastened to a bed on which the rat lies while anesthetized. The fibers (D1–D9) are adjustable, as shown with a phantom. (b) The phantom mimics the anatomy and spectroscopy of rat leg. (c) A rat leg rendering showing the three regions along the diaphysis where Raman spectra were obtained.

Fig. 2

Raman spectra from three different regions of rat leg along the tibial diaphysis. Spectra were normalized to the protein band at $1660{\mathrm{cm}}^{-1}$. (b) Raman spectra from the individual fibers labeled D1–D9 in the wavelength range of 850 to $1100{\mathrm{cm}}^{-1}$. The intensities of the phosphate (bone mineral) band varied with fiber positions. (c) Recovered bone Raman spectra after BTEM was performed on the data in (a) for the three regions. Soft tissue contributions are minimized to recover the bone Raman spectrum. Spectra were normalized to the mineral band at $960{\mathrm{cm}}^{-1}$.

Fig. 3

Transcutaneous Raman spectra acquired from rat leg. The rat leg models were constructed using carbonated hydroxyapatite with varied level of carbonate substitution (0.3%, 4.7%, and 6.9% carbonated hydroxyapatite) as the tibia model. Differences between specimens were captured in the Raman spectra. Intensity of the carbonate band at $1070{\mathrm{cm}}^{-1}$ and the width of the phosphate band at $960{\mathrm{cm}}^{-1}$ both increase with carbonate substitution. The expanded view of the spectra in (a) showing the wavenumber range from 930 to $980{\mathrm{cm}}^{-1}$ is presented as an insert. The position of the ${\mathrm{PO}}_4^{-3}{\nu }_1$ peak shifted from 960 to $958{\mathrm{cm}}^{-1}$ with increasing carbonate substitution.