In-depth confocal Raman microspectrometry (CRM) studies through a planar interface between materials of mismatched refraction indices are known to be affected by a decrease in both collected Raman intensity and axial resolution as a function of focal depth. A complete treatment of these phenomena would require diffraction and refraction effects to be taken into account. Baldwin and Batchelder have recently modeled the refraction effects by considering the influence of the dimensions of the confocal pinhole aperture on the collection efficiency. Their theoretical predictions are compared here with experimental results obtained for a standard 200 µm thick polyethylene (PE) sample. It is shown that the decrease in Raman intensity as a function of focal depth is weaker than predicted, suggesting that off-axis refraction effects cannot be neglected. We therefore propose a simple two-parameter relation which reproduces the observed Raman intensities down to 150-200 µm focal depths. Other in-depth experiments on various test samples, a silicon wafer buried in Nujol oil, PE films of different thicknesses and a polycarbonate slab, were then performed in order to show how the Raman intensity decrease, the radial resolution and the axial interfacial broadening can be directly estimated as a function of the focal depth position. Also, CRM experiments were carried out on a four-layer polymer laminate in order to find the best optical conditions. 'Edge' analyses remain the most efficient way to investigate polymer interfaces but, when in-depth analyses are needed, we tried to evaluate to what extent interfacial broadenings impede the investigation of interpenetration effects at buried interfaces. It is demonstrated that the refraction effects sharpen the interfacial broadenings and the apparent axial resolution does not drastically deteriorate with increasing depth. Indeed, when focused 120 µm deep into a non-absorbing sample, the interface is found to be broadened by a factor of only 7.5 or 3.7 using a 100× or a 50× objective lens; the axial resolution then takes a reasonable value of about 13 µm.

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In this study, the measurement of the true vibrational circular dichroism (VCD) spectrum is considered from an experimental and theoretical approach for any general anisotropic thin solid sample exhibiting linear as well as circular birefringence (LB, CB) and dichroism (LD, CD) properties. For this purpose, we have made use of a simple model alpha-helix polypeptide, namely, the poly(gamma-benzyl-L-glutamate) or PBLG, reference sample possessing a well-known VCD spectrum and giving rise to slightly oriented films by deposition onto a solid substrate. Also, we have used a different Fourier transform infrared modulation of polarization (PM-FTIR) optical setup with two-channel electronic processing in order to record the PM-VLD and PM-VCD spectra for various sample orientations in its film plane. All the corresponding general relations of the expected intensities in these experiments and the related properly designed calibration measurements were established using the Stokes-Mueller formalism; in addition, the residual birefringence of the optical setup and the transmittance anisotropy of the detector were estimated. From a comparative study of the results obtained in solution and in the solid state, we then propose a simple new experimental procedure to extract the true VCD spectrum of an oriented PBLG thin film: its consists of calculating the half-sum of two spectra recorded at theta and at theta +/- 90 degrees sample orientations. Moreover, the complete linear and circular birefringence and dichroism properties of the ordered PBLG thin film are estimated in the amide I and amide II vibrational regions. This allows us to establish for any sample orientation various theoretical simulations of the VCD spectra that agree nicely with the observed experimental results; this confirms that the measurement of LD and LB is in this case a prerequisite in simulating the true VCD spectrum of a partly oriented anisotropic sample. This validates our combined experimental and theoretical approach and opens the route to promising future vibrational CD studies on other macroscopic anisotropic thin film samples.