Measurement of Spatial Resolution and Sensitivity in Transmission and Backscattering Raman Spectroscopy of Opaque Samples: Impact on Pharmaceutical Quality Control and Raman Tomography

Abstract

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.

Effect of Particle Properties of Powders on the Generation and Transmission of Raman Scattering

“…This is likely to be due to differences in the particle shape and/or the absorption properties of the two materials. The attenuation coefficients calculated for Avicel are of a similar magnitude to that estimated from a study by Everall et al 19 A…”

“…Three main observations can be made about the results in Figures 2 and 3: (1) For each powder, the transmitted Raman intensities obtained when the sulphur disc is at the top (P5 in Figures 1a, 2 and 3) or bottom (P1 in Figures 1a, 2 and 3) are similar, but lower than intensities obtained when the disc is within the powder; this arises from photon losses at the air-sample interface when the Raman active disc is at a surface position. 1,[17][18][19] (2) The sulphur Raman signal intensity at the outer and inner (2 mm of powder before the sulphur disc) positions differs by a factor of 1.5, 2.6 and 3.0 for Avicel with a particle size of <38, 53 -106 and 150 -212 µm, respectively. This suggests that a greater portion of photons are lost at the air-sulphur disc interface as the particle size of Avicel increases; larger granular Avicel particles will pack less well than smaller particles and consequently, there are likely to be more air gaps at the Avicel-sulphur disc interface.…”

“…18 It was also shown that use of a photon diode at the sample surfaces can reduce the signal bias towards the centre of the sample and simultaneously improve the signal-to-noise ratio of transmission Raman spectra. 18 From these numerical 1,18 and experimental 1,6,[18][19] A second analyser with a different configuration was also used to investigate the propagation of laser and Raman photons in Avicel with a particle size of 150 -212 m.…”

“…18 However, it can also be seen that the type and size of the powder has a marked effect on the intensity of the transmitted Raman signal from the sulphur inter-layer; the effect of such factors was not investigated in previous studies. 1,[18][19] …”

Variation in the Transmission Near-Infrared Signal with Depth in Turbid Media

“…In comparison, owing to the recent interest in the use of transmission Raman spectrometry for the analysis of pharmaceutical dosage forms, 20,21 there have been a number of theoretical and experimental studies examining the variation of the transmission Raman signal with depth in turbid media. [22][23][24][25][26] It was suggested in a recent paper that the variation in the transmission Raman signal with depth would also be exhibited by transmission NIR spectrometry. 22 While transmission Raman spectrometry can be employed with non-absorbing and weak to moderately absorbing materials, 22 it is inherent to NIR spectrometry that at least weakly absorbing materials are present to give rise to a signal.…”