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

Abstract

Mid-infrared spectra of biological matter such as tissues, or microbial and eukaryotic cells measured in a transmission-type optical setup frequently show strongly distorted line shapes which arise from mixing of absorption and scattering contributions. Scattering-associated distorted line shapes may considerably complicate the analysis and interpretation of the infrared spectra and large efforts have been made to understand the mechanisms of scattering in biological matter and to compensate for spectral alterations caused by scattering. The goals of the present study were twofold: Firstly, to get a deeper understanding of the physics of scattering of biological systems and to explore how physical parameters of the scatterers such as shape, size and refractive index influence the line shape distortions observed. In this context, simulations based on the full Mie scattering formalism for spherical particles were found to be useful to explain the characteristics of the Mie scatter-associated distortions and yielded a size criterion for the scattering particles similar to the well-known near field criterion. The second objective of the study was to investigate whether alternative optical setups allow to minimise the effects of scattering. For this purpose, an optical system is proposed which is composed of an integrating sphere unit originally designed for diffuse reflection measurements, an off-axis DLaTGS detector to collect scattered and transmitted light components and a commercial Fourier transform infrared (FTIR) spectrometer. In the context of this study transmission type (tt-) FTIR spectra and spectra acquired by means of the integrating sphere setup (is-FTIR) were acquired from monodisperse poly(methyl) methacrylate (PMMA) microspheres of systematically varying size. The tt-FTIR spectral data of different PMMA particles confirmed earlier observations such as the presence of size-dependent oscillating spectral baselines, peaks shifts, or derivative-like spectral line shapes. Such effects could be dramatically minimised when is-FTIR spectra were acquired by the integrating sphere unit. Utilisation of an integrating sphere is suggested as a convenient and easy-to use alternative to computer-based methods of scatter correction.

Comparison of cathodic delamination of poly(n‐alkyl methacrylates) on iron

“…The characteristic carbonyl (C = O) stretching mode was found in range of 1731‐1740 cm −1 . In the typical range 2849‐2951 cm −1 the C‐H stretching modes are found . A significant difference in absorbance of the stretching vibration of methylene and methyl group was encountered in the spectra, as expected based on the different alkyl chain lengths of the substituents R, where PSMA shows the strongest peaks, followed by PBMA and PMMA.…”

“…The thickness of the spin‐coated layers was determined by ellipsometry as (950 ± 30) nm, (670 ± 27) nm, and (850 ± 20) nm for PMMA, PBMA, and PSMA, respectively. Furthermore, IR spectra were acquired to examine the composition of polymers on the surface and the positions of the absorption peaks were compared to those from literature . The spectrum of each polymer is displayed in Figure [PMMA, (a); PBMA (b); PSMA, (c)].…”

High throughput absorbance spectra of cancerous cells: a microscopic investigation of spectral artifacts

“…[55][56][57][58][59]. It demonstrates the wide variation of absorbance distribution and Mie scattering effects at microscopic levels and suggests experimental conditions that minimize these problems.…”

Medical Applications of Infrared Spectral Imaging of Individual Cells

“…An ab initio approach to circumvent this problem has been proposed by R. Bhargava (private communication, 2011); but until this approach is implemented in IR microspectrometers, the approximate methods need to be employed. Recently, Dazzi et al [25] demonstrated that the use of an integration sphere for data acquisition can eliminate the problem, but this approach is not suitable for simultaneous detection of multiple pixels of data, using an array detector.…”