Using this probe and a holder with single-fiber illumination, bone Raman spectra were acquired for 60 s at three regions along the diaphysis of the tibia of an anesthetized rat, as shown in Fig. 1(c). This sequence allowed measurement through different thicknesses of overlying soft tissue. The acquired Raman data were processed in MATLAB (MathWorks, Inc., Natick, MA) as previously described to remove cosmic ray spikes, grating-induced curvature, and grating/detector misalignment.20,21 The spectra are reported as wavenumber shifts and are corrected for detector quantum efficiency variation after background subtraction (tissue autofluorescence removal).20,22Figure 2(a) presents unsmoothed Raman spectra from the three diaphysial positions without any removal of overlying tissue contributions. As expected, the intensity of the bone mineral band at relative to collagen protein bands at 1450 and is greater for region 1 (), where the maximum thickness of the overlying soft tissue is about 1 mm. There are increasing contributions from the overlying tissue signal in regions 2 and 3 [ () and () maximum thickness, respectively]. In every case, one or more detectors (fibers) views bone underneath a thin layer of tissue due to the anatomy of the rat leg, so mineral/collagen intensities do not scale linearly with the maximum soft tissue thickness. For example, in region 3, detector fiber 1 is located over the tibia with of soft tissue thickness from the underlying bone, while detectors 2, 3, 4, and 5, and 6, 7, 8, and 9 are more than and , respectively, from the underlying bone. In region 2, fiber 1 is positioned at , fibers 2, 3, 4, and 5 at and fibers 6, 7, 8, and 9 at from the bone. In region 1, all fibers are positioned with approximately the same soft tissue separation () from the underlying bone. The variation in acquired bone mineral Raman signal intensities relative to each individual fiber position is shown in Fig. 2(b) for region 2.