Broadband fiberoptic spectroscopy is investigated for diagnostic applications, based on its ability to noninvasively determine tissue scattering and absorption properties. Spectroscopic instrumentation requires a calibration to account for wavelength dependent factors that may vary, such as the output of the light source, fiberoptic coupling efficiency, ambient light, fiber transmission and detector sensitivity. For techniques such as Diffuse Reflectance Spectroscopy (DRS), a relative calibration of the reflectance is sufficient. For Single Fiber Reflectance spectroscopy (SFR), however, the measured absolute reflectance, R is related to the sample optical properties. Consequently, in order to extract tissue optical properties using SFR, an absolute calibration of the reflectance is required. We investigated two novel SFR calibration methods, using a calibrated mirror and using the Fresnel reflection at the measurement fiber tip as a reference. We compared these to commonly used calibration methods, using either Intralipid-20% in combination with Monte Carlo simulations or Spectralon as a reference. The Fresnel reflection method demonstrated the best reproducibility and yielded the most reliable result. We therefore recommend the Fresnel reflection method for the absolute reflectance calibration of SFR.
To accurately determine sample optical properties using single fiber reflectance spectroscopy (SFR), an absolute calibration of the reflectance is required. We investigated two SFR calibration methods, using a calibrated mirror and using the Fresnel reflection at the fiber tip as a reference. We compared these to commonly used calibration methods, using either Intralipid-20% in combination with Monte Carlo simulations or Spectralon as a reference. The Fresnel reflection method demonstrated the best reproducibility and yielded the most reliable result. We therefore recommend the Fresnel reflection method for the measured absolute reflectance calibration of SFR.
A noninvasive method is introduced for quantification and visualization of fluence rate in light-irradiated biological tissues. The method is based on magnetic resonance thermometry (MRT) measurements of tissue temperature changes resulting from absorption of light. From the spatial–temporal temperature data, the generated heat is calculated. Finally, fluence rate maps are reconstructed by dividing the heat data by the tissue absorption coefficient. Simulations were performed using virtual MRT datasets based on analytically described fluence rate distributions, which could be accurately reconstructed by the method. Next, the approach was tested in gel phantoms. Resulting fluence rate maps matched well with theoretical predictions in a nonscattering phantom (R2=0.93). Experimental validation was further obtained in a scattering phantom, by comparing fluence rates to invasive fluence rate probe measurements along and perpendicular to the optical axis (R2≥0.71 for both cases). Finally, our technique was applied in vivo in a mouse tumor model. The resulting fluence rates matched invasive probe measurements (Pearson’s ρ=0.90, p=0.0026). The method may be applied to investigate the relation between light dose and biological response in light-based treatments, such as photodynamic therapy. It may also be useful for experimental validation of light transport models.
Both Optical Coherence Tomography (OCT) and Single Fiber Reflectance Spectroscopy (SFR) are used to determine various optical properties of tissue. We developed a method combining these two techniques to measure the scattering anisotropy (g1) and γ (=1-g2/1-g1), related to the 1st and 2nd order moments of the phase function. The phase function is intimately associated with the cellular organization and ultrastructure of tissue, physical parameters that may change during disease onset and progression. Quantification of these parameters may therefore allow for improved non-invasive, in vivo discrimination between healthy and diseased tissue.
With SFR the reduced scattering coefficient and γ can be extracted from the reflectance spectrum (Kanick et al., Biomedical Optics Express 2(6), 2011). With OCT the scattering coefficient can be extracted from the signal as a function of depth (Faber et al., Optics Express 12(19), 2004). Consequently, by combining SFR and OCT measurements at the same wavelengths, the scattering anisotropy (g) can be resolved using µs’= µs*(1-g). We performed measurements on a suspension of silica spheres as a proof of principle.
The SFR model for the reflectance as a function of the reduced scattering coefficient and γ is based on semi-empirical modelling. These models feature Monte-Carlo (MC) based model constants. The validity of these constants - and thus the accuracy of the estimated parameters - depends on the phase function employed in the MC simulations. Since the phase function is not known when measuring in tissue, we will investigate the influence of assuming an incorrect phase function on the accuracy of the derived parameters.