In Search of the Microbe/solid Interface: A New Approach Using Super-Resolution Vertical Scanning Interferometry Measurements

The analysis of initial bacterial attachment to solid surfaces is crucial for an improved and quantitative understanding of the development of biofilms on surfaces. Biofilms on rock, metal and glass surfaces play key roles in natural and engineered systems, in diverse processes ranging from weathering and corrosion to charge transfer in microbial fuel cells. Here we present the results of a study using Shewanella oneidensis MR-1 as a model organism. We used Vertical Scanning Interferometry (VSI) and Atomic Force Microscopy (AFM) to investigate the initial stages of cell attachment to glass, steel, aluminum, and carbonate surfaces. Although VSI results obtained using opaque and translucent surfaces are unambiguous, highly-reflective metal surfaces such as polished steel occasionally produce apparent observational artifacts. In these cases, bacteria appear as rod-shaped pits rather than as “positive” cells on the steel surface. This inversion is corrected when the bacteria are treated to increase their opacity. This phenomenon is the result of an interaction between light white light reflected from the bacteria's top membrane surface, and the light reflected from the bacteria-metal interface. These results suggest that (1) information can be recorded from the bacterial cell membrane and bacteria on surfaces using VSI; (2) in addition, with appropriate modifications to the analytical software, these data may offer a unique window for direct study of the bacterial/substrate interface. This optical information, a superposition of at least two correlograms, can thus be used for quantitative observations. In concert with recent progress of super-resolution techniques, imaging and characterization of the previously invisible bacteria-substrate interface in vivo will provide new insights into interactions that occur at this important junction.