Elucidating morphology of bacterial cells from complementary imaging by electron and helium-ion microscopy

• Author(s): Petrovykh, Dmitri Y. ; Silva, A. R. ; Sousa, Cláudia Sofia Cunha ; Exner, Daniela ; Schwaiger, Ruth ; Alves, M. M. ; Pereira, Luciana
• Date: 2021
• Persistent ID: https://hdl.handle.net/1822/75659
• Origin: RepositóriUM - Universidade do Minho
• Document Type: Other
• Language: English
• Contributor(s): Universidade do Minho

Description

When bacterial cells are imaged using vacuum-based high-resolution microscopy techniques, an important consideration is minimizing artifacts, which can arise from sample preparation protocols or from imaging conditions [1,2]. Commonly encountered artifacts include damage to the relatively fragile cells due to sample preparation, vacuum conditions, or high-energy imaging beams. We report on resolving a less common but practically important artifact related to the apparent morphology of cells in scanning electron microscopy (SEM) images. In the standard top-down SEM images, a large fraction of a Vibrio fischeri population appeared as round objects of uniform submicron diameter. As this morphology was unexpected for the investigated V. fischeri bacteria, complementary Helium-ion microscopy (HIM) was employed, taking advantage of its extended depth-of-field [3]. HIM images at off-normal angles revealed that the apparently-round morphology in question was an artifact of top-down imaging of rod-shaped bacteria that became attached perpendicularly to the substrate during the sample preparation. Similar nanoporous polycarbonate membranes are commonly used when preparing bacterial cells for SEM imaging, suggesting that complementary imaging at off-normal angles by SEM or HIM is important for elucidating the true morphology of the investigated cells.