Nano-Investigationof Mineralized Biological SamplesChemical Composition: Experimental Challenges, Constraints, and Considerations

Understanding the chemical composition of calcifications in biological tissues at the nanoscale is crucial for deciphering their formation processes and possible pathological implications. Atomic Force Microscopy Infrared Spectroscopy (AFM-IR), by allowing IR spectroscopy at the nanoscale, is thus a promising strategy to access such highly spatially resolved chemical information. However, these specimens’ inherent morphological and mechanical heterogeneities pose significant challenges for standard resonance-enhanced (RE-AFM-IR) and tapping AFM-IR acquisition modes. This study introduces a dual-mode approach combining tapping and RE-AFM-IR to address these challenges. Tapping AFM-IR is first employed to acquire the topography of the soft and rough surfaces, while RE-AFM-IR provides chemical description at the submicrometric scale through hyperspectral (HS) imaging. This dual-mode methodology is validated on different mineralized biological samples, including breast microcalcifications, revealing the local chemical heterogeneous distribution within the calcium phosphate matrice. Our results outline that dual-mode AFM-IR, coupled with HS imaging, enables robust chemical characterization of highly heterogeneous biomaterials and offers a more comprehensive description compared to conventional AFM-IR single-wavenumber mapping and local spectra.