Laboratoire de Chimie Physique, UMR8000, Université Paris-Sud 11, 91405 Orsay, France.
Analyst. 2013 Jul 21;138(14):4191-201. doi: 10.1039/c3an00381g. Epub 2013 Jun 12.
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 two-fold: 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 in explaining 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 minimisation of 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 sizes. The tt-FTIR spectral data of different PMMA particles confirmed earlier observations such as the presence of size-dependent oscillating spectral baselines, peak 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.
生物物质（如组织或微生物和真核细胞）的中红外光谱在透射式光学设置中测量时，经常会显示出强烈扭曲的线形状，这些线形状是由吸收和散射贡献的混合引起的。与散射相关的扭曲线形状可能会使红外光谱的分析和解释变得非常复杂，因此人们已经做出了很大的努力来理解生物物质中的散射机制，并补偿散射引起的光谱变化。本研究的目的有两个：首先，更深入地了解生物系统散射的物理特性，并探索散射体的物理参数（如形状、大小和折射率）如何影响观察到的线形状扭曲。在这方面，基于球形粒子的全 Mie 散射公式的模拟被发现对于解释 Mie 散射相关的扭曲特征非常有用，并得出了与著名的近场准则类似的散射粒子大小准则。研究的第二个目的是探讨是否可以通过替代光学设置来最小化散射的影响。为此，提出了一种光学系统，该系统由一个积分球单元组成，该单元最初用于漫反射测量，一个离轴 DLaTGS 探测器用于收集散射和透射光分量，以及一台商用傅里叶变换红外（FTIR）光谱仪。在本研究中，从系统变化的大小的单分散聚甲基丙烯酸甲酯（PMMA）微球中获得了透射式（tt-）FTIR 光谱和通过积分球设置获得的光谱（is-FTIR）。不同 PMMA 颗粒的 tt-FTIR 光谱数据证实了早期的观察结果，例如存在与尺寸相关的波动光谱基线、峰位移或导数状光谱线形状。当通过积分球单元获得 is-FTIR 光谱时，可以极大地最小化这些影响。建议使用积分球作为基于计算机的散射校正方法的一种方便易用的替代方法。
