Measurement of spatial resolution and sensitivity in transmission and backscattering Raman spectroscopy of opaque samples: impact on pharmaceutical quality control and Raman tomography.

A practical methodology is described that allows measurement of spatial resolution and sensitivity of Raman spectroscopy in backscatter and transmission modes under conditions where photon migration dominates, i.e., with turbid or opaque samples. For the first time under such conditions the width and intensity of the point spread function (PSF) has been accurately measured as a function of sample thickness and depth below the surface. In transmission mode, the lateral resolution for objects in the bulk degraded linearly with sample thickness, but the resolution was much better for objects near either surface, being determined by the diameter of the probe beam and collection aperture irrespective of sample thickness. In other words, buried objects appear to be larger than ones near either surface. The absolute transmitted signal decreased significantly with sample thickness, but objects in the bulk yielded higher signals than those at either surface. In transmission, materials are sampled preferentially in the bulk, which has ramifications for quantitative analysis. In backscattering mode, objects near the probed surface were detected much more effectively than in the bulk, and the resolution worsened linearly with depth below the surface. These results are highly relevant in circumstances in which one is trying to detect or image buried objects in opaque media, for example Raman tomography of biological tissues or compositional and structural analysis of pharmaceutical tablets. Finally, the observations were in good agreement with Monte Carlo simulations and, provided one is in the diffusion regime, were insensitive to the choice of transport length, which shows that a simple model can be used to predict instrument performance for a given excitation and collection geometry.