Quantitative imaging of individual bacterial cells: E. coli and B. subtilis via terahertz scattering-type scanning near-field optical microscopy

Terahertz (THz) waves have gained significant attention as an imaging technology due to their ability to provide physical and chemical information in a label-free, noninvasive, and nonionizing manner. Notably, their low energy enables nondestructive inspection of internal structures without damaging samples, making them well-suited for biomedical applications. However, the use of THz imaging has been constrained by limited spatial resolution due to the diffraction limit. This study introduces an approach using THz scattering-type scanning near-field optical microscopy, an advanced technique capable of overcoming these limitations and enabling single-cell scale measurements to image and distinguish individual bacterial cells, specifically Escherichia coli and Bacillus subtilis, representing Gram-negative and Gram-positive bacteria, respectively. We utilized tungsten vertical nanoprobes in an apertureless setup to achieve high-resolution imaging. In our experiments, bacteria were measured on a hydrophilic gold substrate with a spatial resolution of 50 nm, demonstrating excellent resolution and image contrast. In addition, quantitative analysis using the line dipole image method allowed calculation of the complex refractive indices, revealing clear differences between the two bacterial species. This technique offers a nonlabel, noninvasive method for bacterial identification, with promising implications for advanced biomedical applications.