The inherent problem of transflection-mode infrared spectroscopic microscopy and the ramifications for biomedical single point and imaging applications

TLDR

Transflection‑mode FTIR spectroscopy is widely used for biomedical samples because it employs inexpensive substrates and a double‑pass path that roughly doubles absorbance. This study presents an optical description of samples placed on multilayer low‑e reflective substrates. By modeling the electric‑field standing wave, the authors explain the non‑linear change in absorbance with sample thickness and its consequences for imaging and classification systems that compare tissues of different thicknesses. They show that distorted spectra can cause classification failures, leading to inaccurate tissue segmentation and potential diagnostic errors.

Abstract

Transflection-mode FTIR spectroscopy has become a popular method of measuring spectra from biomedical and other samples due to the relative low cost of substrates compared to transmission windows, and a higher absorbance due to a double pass through the same sample approximately doubling the effective path length. In this publication we state an optical description of samples on multilayer low-e reflective substrates. Using this model we are able to explain in detail the so-called electric-field standing wave effect and rationalise the non-linear change in absorbance with sample thickness. The ramifications of this non-linear change, for imaging and classification systems, where a model is built from tissue sectioned at a particular thickness and compared with tissue of a different thickness are discussed. We show that spectra can be distorted such that classification fails leading to inaccurate tissue segmentation which may have subsequent implications for disease diagnostics applications.