Development of more effective diagnostic and therapeutic solutions is vital to tackling the growing challenge of bone diseases and disorders in aging societies. Spatially offset Raman spectroscopy (SORS) enables the chemical specificity of conventional Raman spectroscopy to be combined with sub-surface probing. SORS has successfully been applied to transcutaneous investigations of underlying bone and shows great potential to become an The volume within the complex hierarchical bone tissue probed by SORS depends on the specimen's optical properties. Understanding the actual sampling depth is important to correctly assign detected chemical changes to specific areas in the bone. This study explores the hypothesis that the effective Raman signal recovery from certain depths requires different spatial offsets depending on the bone mineralisation. SORS depth investigations were conducted on three bones with significantly different mineralisation levels. Thin slices (0.6 – 1.0 mm thickness) were cut from deer antler, horse metacarpal and whale tympanic bulla and stacked together (4 – 7 layers; 4.1 – 4.7 mm total thickness). A 0.38 mm thin slice of polytetrafluoroethylene (PTFE) served as reference sample and was inserted in between the layers of stacked bone slices. Raman spectra were acquired at 30 s using 830 nm excitation. A quantitative relation between the SORS offset and the primarily interrogated depth inside the bone was established. Maximum accessible depths at small offset strongly depend on the mineralisation level. Using large spatial offsets of 7 – 9 mm PTFE signal recovery depths of 4.4 – 4.6 mm through cortical bone can be realized with only minor dependence on the bone mineralisation. These findings highlight the potential of SORS for medical diagnostics by enabling the non-invasive detection of bone conditions characterised by chemical alterations several millimetres inside compact bone tissue (e.g. infections, tumours, etc.).
In this study we explore the hypothesis that there is a correlation between the ratio of the intensities of specific peaks of the Raman spectrum of bone tissue and the material properties of that particular type of bone. Raman spectroscopy is a powerful analytical technique capable of providing rich chemical information on the composition of skeletal tissue matrices and it has been used extensively to interrogate bone in the past. Spectra are presented of a selection of animal bones, each having greatly differing material properties, the differences having been produced by evolution in response to their greatly differing functions. The main examples described are deer antler (a bone naturally selected for toughness), tympanic bulla from a fin whale (naturally selected for stiffness) and the intermediate ‘standard’ bone from adult mammalian limbs which must be both tough enough to resist fracture and stiff enough to resist deformation during physiological loading (from an ovine femur in our case). In order to illustrate the specific relationship between material properties and Raman spectra additional mineralized tissues also with differing functions and of known Young's moduli are also introduced. The results show that a strong correlation exists between the mineral to collagen ratio of these different bone tissues as measured with Raman spectroscopy and their (previously published) Young's moduli. Raman spectra have been retrieved through skin and tissue in other studies in the past, an amalgamation of refined versions of those in vivo techniques with the work introduced here paves the way for the emergence of novel systems for assessing the material properties of bone tissue at specific anatomical sites in vivo in the future.
