Journal of Biomedical Optics
Raman spectroscopic diffuse tomographic imaging has been demonstrated for the first time. It provides a noninvasive, label-free modality to image the chemical composition of human and animal tissue and other turbid media. This technique has been applied to image the composition of bone tissue within an intact section of a canine limb. Spatially distributed 785-nm laser excitation was employed to prevent thermal damage to the tissue. Diffuse emission tomography reconstruction was used, and the location that was recovered has been confirmed by micro-computed tomography (micro-CT) images.
With recent advances, diffuse tomography shows promise for in vivo clinical imaging.1, 2 In principle, algorithms developed for fluorescence imaging in tissue can be applied to Raman signals. Although the Raman effect is weaker than fluorescence, the scattered signal is detectable, and thus tomography is achievable. Here we demonstrate the first diffuse tomography reconstructions based on Raman scatter.
Raman mapping and imaging are well-established techniques for examining material surfaces.3 Subsurface mapping of simple planar objects was reported recently4, 5 using fiber optic probes with spatially separated injection and collection fibers.6 Noninvasive measurements of bone Raman spectra were demonstrated at depths of5mm" role="presentation">5mm below the skin.5
Bone is promising for Raman tomography because the spectra are rich in compositional information,7 which reflects bone maturity and health. Spectroscopically measured bone composition changes have been correlated with aging8 and susceptibility to osteoporotic fracture.9 The Raman spectrum of bone mineral is easily distinguished from the spectra of proteins and other organic tissue constituents, facilitating recovery of even weak signals by multivariate techniques.
Assessments of bone quantity and quality are essential to detect and monitor fracture risk and fracture healing with disease or injury. Common sites for fracture with osteoporosis are the spine, proximal femur, and distal radius. Stress fractures are most frequently seen in the weight-bearing sites of the tibia and metatarsals. Fracture risk depends on bone geometry, architecture, and material properties, as well as the nature of applied load (magnitude, rate, and direction). As a result, noninvasive imaging and nondestructive analysis methods have been developed to assess many of these bone attributes that are increasingly important to clinical practice and basic research in orthopedics.10 Current clinical in vivo methods include dual-energy x-ray absorptiometry (DXA), quantitative computed tomography (QCT), magnetic resonance imaging (MRI), ultrasound, and most recently, high-resolution peripheral QCT. Ex vivo analyses of bone specimens from patients or animals have also utilized these and other techniques.
In this study, we couple micro-computed tomography (micro-CT) and diffuse optical tomography with Raman spectroscopy to recover spatial and composition information from bone tissue ex vivo. We demonstrate the first reconstruction-based recovery of Raman signals through thick tissues to yield molecular information about subsurface bone tissue. Reconstructions from transcutaneous Raman measurements are challenging, because layers of skin, muscle, fat, and connective tissue lie over the bone sites of interest. These layers have different optical properties and thus variably scatter and polarize the injected light.
We chose a canine model because of specimen availability and a bone size similar to human bone. We selected the tibia, a site that is clinically important and has relatively few overlying soft tissues. Measurements were made on the medial surface, where the only additional optical barrier is the crural extensor retinaculum ligament. The canine hind limb was harvested from an animal euthanized in an approved (UCUCA) University of Michigan study. The section of the limb distal to the knee was excised and scanned using in vivo micro-CT (eXplore Locus RS, GE Healthcare, Ontario, Canada). The tibia was scanned at80kV" role="presentation">80kV and 450μA" role="presentation">450μA with an exposure time of 100ms" role="presentation">100ms using a 360-deg scan technique. The image was reconstructed at a 93-μm" role="presentation">93-μm voxel resolution [Fig. 1a ].
Schulmerich, Matthew V.; Cole, Jacqueline H.; Dooley, Kathryn A.; Morris, Michael D.; Kreider, Jaclynn M.; Goldstein, Steven A.; Srinivasan, Subhadra; and Pogue, Brian W., "Non-Invasive Raman Tomographic Imaging of Canine Bone Tissue" (2008). Open Dartmouth: Faculty Open Access Scholarship. 3632.