The importance of correcting for variable probe-sample interactions in AFM-IR spectroscopy: AFM-IR of dried bacteria...

AFM-IR is a combined atomic force microscopy-infrared spectroscopy method that shows promise for nanoscale chemical characterization of biological-materials interactions. In an effort to apply this method to quantitatively probe mechanisms of microbiologically induced polyurethane degradation, we have investigated monolayer clusters of ∼200 nm thick Pseudomonas protegens Pf-5 bacteria (Pf) on a 300 nm thick polyether-polyurethane (PU) film. Here, the impact of the different biological and polymer mechanical properties on the thermomechanical AFM-IR detection mechanism was first assessed without the additional complication of polymer degradation. AFM-IR spectra of Pf and PU were compared with FTIR and showed good agreement. Local AFM-IR spectra of Pf on PU (Pf-PU) exhibited bands from both constituents, showing that AFM-IR is sensitive to chemical composition both at and below the surface. One distinct difference in local AFM-IR spectra on Pf-PU was an anomalous ∼4× increase in IR peak intensities for the probe in contact with Pf versus PU. This was attributed to differences in probe-sample interactions. In particular, significantly higher cantilever damping was observed for probe contact with PU, with a ∼10× smaller Q factor. AFM-IR chemical mapping at single wavelengths was also affected. We demonstrate ratioing of mapping data for chemical analysis as a simple method to cancel the extreme effects of the variable probe-sample interactions.