The importance of correcting for variable probe–sample interactions in AFM-IR spectroscopy: AFM-IR of dried bacteria on a polyurethane film

Abstract

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.

Dynamic structure and composition of bone investigated by nanoscale infrared spectroscopy

“…Good agreement was shown previously between AFM-IR and FTIR spectra on polymers [33,44], but the ratios appeared to be different for heterogeneous materials [42], most likely due to scale effects. FTIR measures absorption from the bulk, whereas AFM-IR permits local measurement at higher resolution.…”

Characterization of Intact Eukaryotic Cells with Subcellular Spatial Resolution by Photothermal-Induced Resonance Infrared Spectroscopy and Imaging

“…[24] However, complications are observed for layered materials, where the mechanical properties of the tip-sample contact affect signal intensity. [25] In our experiments, the linearity of the response with thickness is confirmed when looking at nuclei of different thickness (See Supporting Information and Supporting Figure 1). However, the relationship between sample thickness and signal intensity is no longer linear when comparing different subcellular regions.…”

Probe–Sample Interaction-Independent Atomic Force Microscopy–Infrared Spectroscopy: Toward Robust Nanoscale Compositional Mapping

“…13, 14 At present, however, this imaging modality suffers from signal fluctuations resulting from probe-sample mechanical interactions. 15 These fluctuations can have little or no correlation to the local sample expansion (or spectral contrast). It has been shown that these fluctuations can be mitigated by tracking a cantilever resonance peak 16 during data acquisition (hereafter referred to as resonance enhanced operation) or by using IR peak ratios 15 for analysis post-acquisition.…”

“…15 These fluctuations can have little or no correlation to the local sample expansion (or spectral contrast). It has been shown that these fluctuations can be mitigated by tracking a cantilever resonance peak 16 during data acquisition (hereafter referred to as resonance enhanced operation) or by using IR peak ratios 15 for analysis post-acquisition. These methods, however, restrict the available data and are not always effective.…”