Liquid Phase Backscattered Scanning Electron Microscopy of Bacillus subtilis Spores

Backscattered electron scanning electron microscopy (BSE-SEM) provides compositional image contrast but has found limited application to biological samples due to the low atomic number difference between constituent elements, the thickness of the surrounding environment, and the need for complex sample preparation. Previous graphene-encapsulation SEM studies have demonstrated imaging of hydrated bacteria, principally to preserve or observe bacterial morphology. Here, we establish liquid phase BSE-SEM (LPBSEM) as a depth-sensitive, label-free approach for visualizing subsurface architecture in intact hydrated bacteria and Bacillus subtilis spores. This approach does not require staining and enables high-contrast visualization of subcellular structures in hydrated samples. Internal structural layers within B. subtilis spores have been observed similar to those visualized with conventional thin-section transmission electron microscopy but without the need for sample preparation that potentially compromises sample integrity. We further investigate the influence of beam energy on the interaction volume depth and image contrast and propose optimal conditions for subsurface visualization. Monte Carlo simulations were used to assess whether the observed contrast trends are consistent with expected backscatter distributions and provide a physical framework for interpretation and understanding of BSE generation from hydrated, low atomic number specimens.