Dept. of Biomedical Engineering
Oregon Health & Science University
Optical techniques represent a non-invasive strategy to monitor normal and pathological tissue function in both diagnostic and therapeutic applications. Central to the utility of optical methods in medicine is the determination of how light propagates in tissues. This requires a knowledge of light-tissue interactions which are determined by scattering and absorption. Scattering is dependent on the nanoscale organization of the tissue: given by the size, shape and density of the tissue constituents. Scattering is quantified by two properties: the number of scattering events encountered by photons per unit distance, termed the scattering coefficient, denoted Î¼[subscript s] [cm superscript -1]; and the cosine of the average scattering angle during each scattering event, termed the anisotropy factor, denoted g [dimensionless]. For a fixed wavelength of light, the Î¼[subscript s] is sensitive to both the density, shape and size of the constituents of the tissue, while the g is most sensitive to constituent size. A complete knowledge of tissue structure requires the simultaneous knowledge of both these optical properties. Unfortunately, current techniques available to measure the scattering properties of a tissue report a quantity that couples these parameters, termed the reduced scattering coefficient, denoted Î¼[subscript s] â[prime] and equal to Î¼[subscript s] (1-g). Hence, constituent size and density of a tissue cannot be inferred through such measurements. This thesis presents a technique to simultaneously estimate the scattering co-efficient and anisotropy of tissues using a reflectance-mode confocal scanning laser microscope (rCSLM). The rCSLM signal is fit to a two parameter decaying exponential to yield two empirically fit parameters: the attenuation Î¼ [cm[superscript -1]], and reflectivity Ï[-]. A theoretical model was developed to map these empirical fit parameters to the optical properties of the scattering media: Î¼[subscript s] and g. This model was validated on microsphere suspensions, whose optical properties are calculable from first principles using well established electromagnetic scattering calculations, known as Mie theory. Variable concentrations and sizes of micro-spheres were tested. The optical properties of the sphere suspensions determined from the rCSLM data were found to agree with the corresponding Mie theoretic values. The model is used to estimate optical properties of different types of mouse tissue. Next, to demonstrate the utility of the technique, the optical clearing effect in dermal tissue was investigated. Optical clearing consists of exposing tissues to chemical agents, such as glycerin, that make tissues appear semi-transparent. Whether the increased transparency is due to increases in scattering anisotropyor a reduction in the scattering coefficient has been ill-defined. Our simultaneous measurement of both the scattering coefficient and anisotropy factor using rCSLM data revealed that glycerin significantly increased the g of the dermis from 0.7 to0.99, with little change in Î¼[subscript s] of the dermis. These results indicate that glycerin increases the size of the scattering constituents in the dermis resulting in increased transparency of the tissue. Lastly, the method was employed in a pilot study to characterize the structural consequences of osteogenesis imperfecta (oim), a genetic disorder that affects the ability of collagen fibers to organize into fiber bundles within the dermis. Mice with and without the oim mutation were investigated. The scattering anisotropy g decreased from 0.81 Â±0.1 in wild type mice to 0.46 Â±0.2 in mice with the mutation.The scattering coefficient was determined to be 70 Â±20 cm[superscript -1] in wild type miceand 90 Â±30 cm[superscript -1] in oim mutated mice. The decrease in g, provides an optically derived indication of the failure of the mutant collagen fibrils to assemble into larger collagen fiber bundles present in the dermis of wild type mice. While preliminary, these results demonstrate the potential of the rCSLM based method to identify structural changes in tissues due to pathological conditions. Together, these studies indicate the unique utility of rCSLM to perform non-invasive measurements of tissue structure through the simultaneous measurement of the scattering coefficient and scattering anisotropy factor using the model presented in this thesis. Development of pathology in tissues is associated with structural changes that can be monitored non-invasively by tracking the changes in optical properties of tissue.
School of Medicine
Samatham Venkata, Ravikant, "Determination of optical scattering properties of tissues using reflectance-mode confocal microscopy" (2012). Scholar Archive. 861.