Confocal Raman microspectroscopy is a relevant and useful tool to perform in vivo diagnosis of cutaneous tissues noninvasively and without labeling. This optical technique provides in-depth molecular and conformational characterization of skin. Unfortunately, spectral distortions occur due to elastic scattering. Our objective is to correct the attenuation of in-depth Raman peaks intensity by considering elastic scattering in biological tissues. In this purpose, a correction model was constructed using skin scattering properties as parameters thus enabling quantitative analysis. The work presented here is a technique of in vivo Diffuse Reflectance Micro-Spectroscopy called Micro-DRS. It achieves optical properties characterization in the skin layers probed by Raman microspectroscopy. The Micro-DRS setup can easily be coupled to a confocal Raman micro-probe to perform simultaneous measurements. Thanks to Monte Carlo simulations and experimental results obtained on homemade solid phantoms mimicking skin optical properties, we show that it is possible to measure the absorption coefficient μa, the reduced scattering coefficient μs', the scattering coefficient μs and the anisotropy of scattering g with this new apparatus. The measured scattering properties can be used subsequently as parameters in our correction model. Coupled to a Raman micro-spectrometer, Micro-DRS enables a quantitative analysis when tracking drug penetration through skin and it can be used independently to provide additional diagnosing criterions.
KEYWORDS: Optical transfer functions, Optical properties, Imaging systems, Scattering, Spectroscopy, Monte Carlo methods, Algorithm development, Signal detection, Electroluminescent displays, Absorption
We validate a non-contact Diffuse Reflectance Spectroscopy (DRS) system as a first stage to approach quantitative multi-spectral imaging technique. The non-contact DRS system with separated illumination and detection paths was developed with different progressive set-ups which were all compared to a well-founded contact DRS system. While quantitation of the absorption coefficient is well achieved with the existing method, the calculation of the scattering coefficient is deteriorated by the non-contact architecture measurements. We have therefore developed an adaptive reference-based algorithm to compensate for this effect.
Diffuse reflectance spectroscopy characterizes composition and structure of tissues by determining their scattering and absorption properties. We have developed in our laboratory a low-cost spatially resolved diffuse reflectance spectroscopy instrument. We present in this study some results showing how to adapt this technology on multi-layered tissues. First of all, a method enabling determination of scattering and absorption properties of multi-layered phantoms is described; the adaptation of the initial methodology to focus on deep layers is especially detailed. Then some preliminary results obtained on a panel of volunteer’s redness faces are presented.
Light/tissue interactions, like diffuse reflectance, endogenous fluorescence and Raman scattering, are a powerful means for providing skin diagnosis. Instrument calibration is an important step. We thus developed multilayered phantoms for calibration of optical systems. These phantoms mimic the optical properties of biological tissues such as skin. Our final objective is to better understand light/tissue interactions especially in the case of confocal Raman spectroscopy.
The phantom preparation procedure is described, including the employed method to obtain a stratified object. PDMS was chosen as the bulk material. TiO2 was used as light scattering agent. Dye and ink were adopted to mimic, respectively, oxy-hemoglobin and melanin absorption spectra. By varying the amount of the incorporated components, we created a material with tunable optical properties.
Monolayer and multilayered phantoms were designed to allow several characterization methods. Among them, we can name: X-ray tomography for structural information; Diffuse Reflectance Spectroscopy (DRS) with a homemade fibered bundle system for optical characterization; and Raman depth profiling with a commercial confocal Raman microscope for structural information and for our final objective.
For each technique, the obtained results are presented and correlated when possible.
A few words are said on our final objective. Raman depth profiles of the multilayered phantoms are distorted by elastic scattering. The signal attenuation through each single layer is directly dependent on its own scattering property. Therefore, determining the optical properties, obtained here with DRS, is crucial to properly correct Raman depth profiles. Thus, it would be permitted to consider quantitative studies on skin for drug permeation follow-up or hydration assessment, for instance.
Confocal Raman microspectroscopy allows in-depth molecular and conformational characterization of biological tissues non-invasively. Unfortunately, spectral distortions occur due to elastic scattering. Our objective is to correct the attenuation of in-depth Raman peaks intensity by considering this phenomenon, enabling thus quantitative diagnosis. In this purpose, we developed PDMS phantoms mimicking skin optical properties used as tools for instrument calibration and data processing method validation. An optical system based on a fibers bundle has been previously developed for in vivo skin characterization with Diffuse Reflectance Spectroscopy (DRS). Used on our phantoms, this technique allows checking their optical properties: the targeted ones were retrieved. Raman microspectroscopy was performed using a commercial confocal microscope. Depth profiles were constructed from integrated intensity of some specific PDMS Raman vibrations. Acquired on monolayer phantoms, they display a decline which is increasing with the scattering coefficient. Furthermore, when acquiring Raman spectra on multilayered phantoms, the signal attenuation through each single layer is directly dependent on its own scattering property. Therefore, determining the optical properties of any biological sample, obtained with DRS for example, is crucial to correct properly Raman depth profiles. A model, inspired from S.L. Jacques's expression for Confocal Reflectance Microscopy and modified at some points, is proposed and tested to fit the depth profiles obtained on the phantoms as function of the reduced scattering coefficient. Consequently, once the optical properties of a biological sample are known, the intensity of deep Raman spectra distorted by elastic scattering can be corrected with our reliable model, permitting thus to consider quantitative studies for purposes of characterization or diagnosis.