Optical standing-wave artifacts in reflection-absorption FTIR microspectroscopy of biological materials

Reflection-absorption spectra obtained with an infrared microscope should yield the same absorption coefficients as direct micro-transmission measurements as long as the correct effective sample thickness is used, but in practice, severe optical artifacts can complicate the spectra. Using deposited protein gel fdms as a homogenous model for biological cell-like samples, we demonstrate the effect of standing-wave interference of the IR beam at the reflective substrate surface which dramatically and systematically alters the absorbance intensity across the spectrum as a function of sample thickness. To explain the observed spectral artifacts, we simulate the optical standing-wave for the focussed IR beam, and insert the parameters into an existing standing-wave absorption theory. By introducing an additional term to the theory representing a component of the standing-wave resonant with the film thickness, the data are accurately reproduced, and the relative band intensities can be corrected to the direct transmission values. This approach may be generally applicable in reflection-absorption experiments to obtain reliable absorbance spectra of homogenous samples even when the sample thickness is larger than the IR wavelength.