Spatially offset Raman spectroscopy (SORS) is currently being developed as an
in vivo tool for bone disease detection, but to date, information about the interrogated volume as influenced by the light propagation and scattering characteristics of the bone matrix is still limited. This paper seeks to develop our general understanding of the sampling depths of SORS in bone specimens as a function of the applied spatial offset. Equine metacarpal bone was selected as a suitable specimen of compact cortical bone large enough to allow several thin slices (600 μm) to be cut from the dorsal surface. Photon migration at 830-nm excitation was studied with five bone slices and a 380 μm-thin polytetrafluoroethylene (PTFE) slice placed consecutively between the layers. To optimize Raman signal recovery of the PTFE with increasing depth within the bone stack required a corresponding increase in spatial offset. For example, to sample effectively at 2.2-mm depth within the bone required an optimal SORS offset of 7mm. However, with a 7-mm offset, the maximum accessible penetration depth from which the PTFE signal could be still recovered was 3.7mm. These results provide essential basic information for developing SORS technology for medical diagnostics in general and optimizing sampling through bone tissue, permitting a better understanding of the relationship between the offset and depth of bone assessed, in particular. Potential applications include the detection of chemically specific markers for changes in bone matrix chemistry localized within the tissue and not present in healthy bone.