Resonant Mie scattering in infrared spectroscopy of biological materials – understanding the ‘dispersion artefact’

Abstract

Infrared spectroscopic cytology is potentially a powerful clinical tool. However, in order for it to be successful, practitioners must be able to extract reliably a pure absorption spectrum from a measured spectrum that often contains many confounding factors. The most intractable problem to date is the, so called, dispersion artefact which most prominently manifests itself as a sharp decrease in absorbance on the high wavenumber side of the amide I band in the measured spectrum, exhibiting a derivative-like line shape. In this paper we use synchrotron radiation FTIR micro-spectroscopy to record spectra of mono-dispersed poly(methyl methacrylate) (PMMA) spheres of systematically varying size and demonstrate that the spectral distortions in the data can be understood in terms of resonant Mie scattering. A full understanding of this effect will enable us to develop strategies for deconvolving the scattering contribution and recovering the pure absorption spectrum, thus removing one of the last technological barriers to the development of clinical spectroscopic cytology.

Minimising contributions from scattering in infrared spectra by means of an integrating sphere

“…Furthermore, the shape of the carbonyl band has become asymmetric with a negative side lobe at the short wavelength side. These spectral changes are in good agreement with earlier observations of Bassan et al 17 , who found similar changes in IR microspectra of individual PMMA microspheres. Contrary to the findings, the is-FTIR spectrum showed no significant differences compared to the pure absorption spectrum of Fig.…”

“…7,8,[15][16][17][18] Typical spectral characteristics of Mie scattering are broad baseline effects (oscillating baselines, baseline slopes) and sharp scattering features (e.g. derivativelike line shapes, band shifts, inverted bands, etc.).…”

“…Because many biomedical applications of IR spectroscopy rely on observing specific disease-associated spectral markers or signatures, scattering renders the analysis of the spectral data very difficult if not impossible. 7,8,[15][16][17][18] IR spectral scattering effects are manifested in a typical manner and express themselves as altered spectral baselines (baseline slopes, undulating baselines), shifted peak positions, derivative-like band profiles, or may even lead to inverted (negative) peak shapes. 15,17 Many authors have stated that spectra from biomedical samples such as cells and tissues can be influenced by factors other than the biochemical composition.…”

“…7,8,[15][16][17][18] IR spectral scattering effects are manifested in a typical manner and express themselves as altered spectral baselines (baseline slopes, undulating baselines), shifted peak positions, derivative-like band profiles, or may even lead to inverted (negative) peak shapes. 15,17 Many authors have stated that spectra from biomedical samples such as cells and tissues can be influenced by factors other than the biochemical composition. 18 For example, Mohlenhoff et al reported that the nuclei of eukaryotic cells act as scatterers causing a broad, undulating spectral background onto which the absorption features are superimposed.…”

“…in resonance) prompted the authors to term this optical process Resonant Mie Scattering (RMieS). 17 Based on this finding, the same group developed in the following years theoretical models of scattering which could be incorporated into computer algorithms to minimise unwanted scattering contributions from IR spectra showing both, broad Mie-type scattering features and spectral characteristics caused by RMieS. 8,22 Although with the described computer models tremendous progress has been achieved, it must be noted that scattering effects cannot be always completely eliminated from the experimental data.…”