Light Signal Attenuation by Murine Cortical Bone for Quantification of Fluorescence Reflectance Imaging

Fluorescence reflectance imaging is a powerful technique to study microscopic processes on a microscopic scale in bio-molecular research. This enables for example in vivo-monitoring of metabolic and oncologic processes in bone tissue of small animals. However, the detection of light signals originating from biological structures labelled with fluorescent dyes is subject to attenuation processes occuring in the surrounding tissue. This introduces an unwanted bias, which has already been quantified in many previous studies in order to correct the measured light signal for attenuation. It was thus found that the attenuation does not only depend on the tissue type under examination but on the specific set up of the experiment as well. It was therefore the aim of this project to quantify light signal attenuation in bone tissue as a function of cortical thickness (d) and signal wavelength (λ) to assess the feasibility to correct for attenuation effects in molecular imaging of small animals employing a commonly used experimental set up including fluorescence reflectance imaging. Bone samples were prepared from diaphyses of murine femora and tibiae. Micro-computed tomography was then used to determine the cortical thickness and the tissue mineral density of these samples, which were then used to attenuate well defined light signals produced by light emitting diodes. These were chosen to match the absorption and emission spectra of commonly used fluorescent dyes (from λ = 470 nm to λ = 820 nm). Fluorescence reflectance imaging was used to measure the attenuated signals. An exponential decay of the light signal with increasing cortical thickness (from d = 126 μm to d = 239 μm) was observed. The attenuation was quantified with the reduced scattering coefficient. This parameter was derived from a multivariate regression model that was based on the diffusion approximation to the transport theory. But it was not possible to resolve the wavelength-dependence of the reduced scattering coefficient within the chosen wavelength spectrum. A second study with a modified set up yielded qualitatively similar data with a reduced scattering coefficient which showed a significant difference compared to the corresponding coefficient of the main experimental study. It was thus also confirmed with the current project that the acquired data is substantially affected by the specifics of the experimental set up. The results of this project show that a correction for attenuation effects using optical data from fluorescence reflectance imaging is feasible when combined with another imaging modality, such as micro-computed tomography. Using only fluorescence reflectance imaging in a uni-modal approach, such as ratiometry, to correct attenuated light signals is not feasible with the current experimental set up due to the limited wavelength-dependence of the derived optical bone tissue properties. This project also clarified the importance of the specific experimental set up. It is therefore strongly suggested to perform calibration measurements prior to future bio-molecular studies trying to correct for tissue attenuation in order to obtain quantitative results from fluorescent reflectance imaging, such as the fluorescent dye concentration. Both experimental set ups should be as similar to each other as possible to obtain reliable data.


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