Optical artefacts in transflection mode FTIR microspectroscopic images of single cells on a biological support: the effect of back-scattering into collection optics.

Infrared microspectroscopic imaging data of single human prostate cancer cells, on an artificial extracellular matrix (Matrigel) thin-film surface, are presented. The spectral intensity maps, obtained in reflection mode, appear to show that the protein intensity distribution observed at the location of a cell changes dramatically depending on the concentration and/or thickness of the underlying Matrigel layer. Specifically, cells adhered to a low protein concentration or thin surface exhibit a higher protein intensity signal than the surrounding layer whereas those on a high protein concentration or thick surface exhibit a lower protein intensity signal. These results are qualitatively explained by a simple model that takes into account the fact that radiation scattered from cells can enter the collection optics of the microscope without passing through the Matrigel layer. This leads to an apparent reduction in absorption at the cell.