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AFM-IR combines the chemical sensitivity of infrared spectroscopy with the lateral resolution of scanning probe microscopy, allowing nanoscale chemical analysis of almost any organic material under ambient conditions. As a result, this versatile technique is rapidly gaining popularity among materials scientists. Here, we report a previously overlooked source of data and artifacts in AFM-IR analysis; reflection from the buried interface. Periodic arrays of gold on glass are used to show that the overall signal in AFM-IR is affected by the wavelength-dependent reflectivity and thermal response of the underlying substrate. Excitingly, this demonstrates that remote analysis of heterogeneities at the buried interface is possible alongside that of an overlying organic film. On the other hand, AFM-IR users should carefully consider the composition and topography of underlying substrates when interpreting nanoscale infrared data. The common practice of generating ratio images, or indeed the normalization of AFM-IR spectra, should be approached with caution in the presence of substrate heterogeneity or variable sample thickness.

Reflectance in AFM-IR: Implications for Interpretation and Remote Analysis of the Buried Interface