Nanochemical Cell-Surface Evaluation in Photothermal Spectroscopic Imaging of Antimicrobial Interactions in the Model...

The power of photothermal spectroscopic imaging to visualize antimicrobial interactions on the surfaces of individual bacteria cells has been demonstrated on the model system Bacillus subtilis 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. [J. Phys. Chem. C 2025, 129, 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 B. subtilis 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 B. subtilis 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.