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. 2009 Mar;63(3):286-95.
doi: 10.1366/000370209787599013.

Transcutaneous Raman spectroscopy of murine bone in vivo

Affiliations

Transcutaneous Raman spectroscopy of murine bone in vivo

Matthew V Schulmerich et al. Appl Spectrosc. 2009 Mar.

Abstract

Raman spectroscopy can provide valuable information about bone tissue composition in studies of bone development, biomechanics, and health. In order to study the Raman spectra of bone in vivo, instrumentation that enhances the recovery of subsurface spectra must be developed and validated. Five fiber-optic probe configurations were considered for transcutaneous bone Raman spectroscopy of small animals. Measurements were obtained from the tibia of sacrificed mice, and the bone Raman signal was recovered for each probe configuration. The configuration with the optimal combination of bone signal intensity, signal variance, and power distribution was then evaluated under in vivo conditions. Multiple in vivo transcutaneous measurements were obtained from the left tibia of 32 anesthetized mice. After collecting the transcutaneous Raman signal, exposed bone measurements were collected and used as a validation reference. Multivariate analysis was used to recover bone spectra from transcutaneous measurements. To assess the validity of the transcutaneous bone measurements cross-correlations were calculated between standardized spectra from the recovered bone signal and the exposed bone measurements. Additionally, the carbonate-to-phosphate height ratios of the recovered bone signals were compared to the reference exposed bone measurements. The mean cross-correlation coefficient between the recovered and exposed measurements was 0.96, and the carbonate-to-phosphate ratios did not differ significantly between the two sets of spectra (p > 0.05). During these first systematic in vivo Raman measurements, we discovered that probe alignment and animal coat color influenced the results and thus should be considered in future probe and study designs. Nevertheless, our noninvasive Raman spectroscopic probe accurately assessed bone tissue composition through the skin in live mice.

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Figures

Fig. 1
Fig. 1
Schematic depicting spatially offset Raman spectroscopy. The spatial separation between illumination and collection optics allows depth probing into specimens. Collection optics located at a small distance from the illumination source (d1) will collect Raman scattered light originating in regions near the specimen surface, while collection optics located at a greater distance from the illumination source (d2) will collect scatter originating in deeper layers. The stars represent the points where Raman scattered photons are emitted.
Fig. 2
Fig. 2
(a) Schematic of Raman spectroscopy system for in vivo measurements on mice. (b) Photograph of mouse restraint, showing water cooling and anesthesia delivery subsystems.
Fig. 3
Fig. 3
Schematic of a mouse tibia with five different configurations of the illumination (striped) and collection (solid) regions.
Fig. 4
Fig. 4
Schematic of probe alignment on a left mouse tibia, which differed according to the limb curvature.
Fig. 5
Fig. 5
(a) Mean transcutaneous signal taken from the 50 collection fibers for the five different Raman probe configurations of Fig. 3. (b) Superimposed standardized spectra from each of the fifty collection fibers for each probe configuration.
Fig. 6
Fig. 6
Representative recovered bone factor for the in vivo transcutaneous measurements (a) compared to the individual exposed bone spectra and (b) the mean exposed bone spectrum of specimen 19. (c) The recovered bone factors were similar to the mean exposed bone spectra, as evidenced by the high cross-correlation coefficients between the two for all specimens.
Fig. 7
Fig. 7
(a) Mean carbonate-to-phosphate ratio of the recovered bone factor and exposed bone measurements for all mice. (b) Mean carbonate-to-phosphate ratio of the bone factor and exposed measurements for each mouse. Error bars denote standard deviation for both plots.
Fig. 8
Fig. 8
(a) Cross-correlation coefficient between the recovered bone factors and the mean exposed bone spectra, grouped by coat color. (b) Coefficient of variation in the cross-correlation coefficients, grouped by coat color. Values are mean ± standard deviation.
Fig. 9
Fig. 9
Phosphate-to-phenylalanine band height ratio taken from the transcutaneous signal collected by each fiber for each of the probe alignments. Values are mean ± standard deviation.
Fig. 10
Fig. 10
(a) Difference in the carbonate-to-phosphate ratio between the recovered bone factor and the exposed bone spectrum for each specimen and mean difference over all four mice (dashed line) for each probe alignment (b) Coefficient of variation between the carbonate-to-phosphate ratios for each of the probe alignments. Values are mean ± standard deviation.

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