Nanochemical Cell-Surface Evaluation in Photothermal Spectroscopic Imaging of Antimicrobial Interactions in the Model System <i>Bacillus subtilis</i> and Vancomycin

The power of photothermal spectroscopic imaging to visualize antimicrobial interactions on the surfaces of individual bacteria cells has been demonstrated on the model system <i>Bacillus subtilis</i> and vancomycin using mid-infrared photoinduced force microscopy (PiF-IR, also mid-IR PiFM). High-resolution PiF contrasts obtained by merging subsequent PiF-IR scans at two different illumination frequencies revealed chemical details of cell wall destruction after 30 and 60 min incubation with vancomycin with a spatial resolution of ∼5 nm. This approach compensates for local intensity variations induced by near-field coupling of the illuminating electric field with nanostructured surfaces, which appear in single-frequency contrasts in photothermal imaging methods, as shown by Anindo et al. [<i>J. Phys. Chem. C</i> <b>2025</b>, <i>129</i>, 4517. DOI: 10.1021/acs.jpcc.4c08305]. Known spectral shifts associated with hydrogen bond formation between vancomycin and the N-acyl-d-Ala₄-d-Ala₅ termini in the peptidoglycan cell wall have been observed in chemometrics of PiF-IR spectra from treated and untreated <i>B. subtilis</i> harvested after 30 min from the same experiment. Spectral signatures of the vancomyin interaction have been located in the piecrust of a progressing septum with ∼10 nm resolution using PiF contrasts of three selected bands of a PiF-IR hyperspectral scan of an individual <i>B. subtilis</i> cell harvested after 30 min incubation. Our results are complemented by a discussion of imaging artifacts and the influence of parameter settings supporting further development toward standardization in the application of PiF-IR for visualizing the chemical interaction of antibiotics on the surface of microbes with few nanometer resolution.