Subsurface probing in diffusely scattering media using spatially offset Raman spectroscopy.

We describe a simple methodology for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The technique is based on the collection of Raman scattered light from surface regions that are laterally offset away from the excitation laser spot on the sample. The Raman spectra obtained in this way exhibit a variation in relative spectral intensities of the surface and subsurface layers of the sample being investigated. The data set is processed using a multivariate data analysis to yield pure Raman spectra of the individual sample layers, providing a method for the effective elimination of surface Raman scatter. The methodology is applicable to the retrieval of pure Raman spectra from depths well in excess of those accessible with conventional confocal microscopy. In this first feasibility study we have differentiated between surface and subsurface Raman signals within a diffusely scattering sample composed of two layers: trans-stilbene powder beneath a 1 mm thick over-layer of PMMA (poly(methyl methacrylate)) powder. The improvement in contrast of the subsurface trans-stilbene layer without numerical processing was 19 times. The potential applications include biomedical subsurface probing of specific tissues through different overlying tissues such as assessment of bone quality through skin, providing an effective noninvasive means of screening for bone degeneration, other skeletal disease diagnosis, and dermatology studies, as well as materials and catalyst research.

Depth profiling in diffusely scattering media using Raman spectroscopy and picosecond Kerr gating.

We demonstrate how pulsed laser Raman excitation (approximately 1 ps) followed by fast optical Kerr gating (approximately 4 ps) can be used to effectively separate Raman signals originating from different depths in heterogeneous diffusely scattering media. The diffuse scattering slows down photon propagation through turbid samples enabling higher depth resolution than would be obtained for a given instrumental time resolution in an optically transparent medium. Two types of experiments on two-layer systems demonstrate the ability to differentiate between surface and sub-surface Raman signals. A Raman spectrum was obtained of stilbene powder buried beneath a 1 mm over-layer of PMMA (poly(methyl methacrylate)) powder. The signal contrasts of the lower stilbene layer and upper PMMA layer were improved by factors >or=5 and >or=180, respectively, by rejecting the Raman component of the counterpart layer. The ability to select the Raman signal of a thin top surface layer in preference to those from an underlying diffusely scattering substrate was demonstrated using a 100 mum thick optically transparent film of PET (poly(ethylene terephthalate)) on top of stilbene powder. The gating resulted in the suppression of the underlying stilbene Raman signal by a factor of 1200. The experiments were performed in back-scattering geometry using 400 nm excitation wavelength. The experimental technique should be well suited to biomedical applications such as disease diagnosis.

Numerical simulations of subsurface probing in diffusely scattering media using spatially offset Raman spectroscopy.

We present the first elementary model predicting how Raman intensities vary for a range of experimental variables for spatially offset Raman spectroscopy (SORS), a recently proposed technique for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The model was able to reproduce the key observations made from the first SORS experiments, namely the dependence of Raman signal intensities on the spatial offset between the illumination and collection points and the relative contributions to the overall spectrum from the top layer and sub-layer. The application of the SORS concept to a three-layer system is also discussed. The model also clearly indicates that an annular geometry, rather than a point-collection geometry, which was used in the earlier experiments, would yield much improved data.

Analysis of thin film coatings on poly(ethylene terephthalate) by confocal Raman microscopy and surface-enhanced Raman scattering.

The characterization of thin coatings on polymers such as poly(ethylene terephthalate) (PET) is required in order to study chemical composition and coating continuity. Two different methods of applying Raman spectroscopy for this purpose are compared in this paper. Using confocal Raman microscopy, thick coatings (> 10 microns) are relatively easily identified; however, the Raman scattering from the acrylic coatings commonly used is much weaker than that of PET and consequently, there is a background due to the substrate. Thin acrylic coatings (< 1 micron) usually cannot be detected. Surface-enhanced Raman scattering (SERS) of uncoated PET gives intense signals and if the spectra are taken from the metal-coated side, there is no evidence of the underlying Raman scattering from the bulk. Acrylic coatings do not give sufficiently strong or reproducible SERS to be reliably identified, but even thin (20 nm) coatings completely block the SERS from the substrate. Only where gaps appear in the coating is the SERS of the underlying PET seen. To detect a positive signal from the coating, SERS active labels were incorporated into the acrylic at low concentrations either as a physical mixture or as reactive co-monomers. This uniquely labels the coating and allows detection and, in principle, mapping of the coverage. Thus, for thick (> > 1 micron) coatings, normal Raman spectroscopy is an effective technique for detecting the presence of the surface coating. However, it is ineffective with thin (< 1 micron) coatings, and SERS alone only indicates where the coating is incomplete or defective. However, when a SERS label is added, spectra can be detected from very thin coatings (20 nm). The concentration of the labels is sufficiently low for the coating to remain colorless.