To understand the development of biofilms on metal surfaces, analysis of initial bacterial attachment to surfaces is crucial. Here we present the results of a study, using Shewanella oneidensis MR-1 as a model organism, in which vertical scanning interferometry (VSI) was used to investigate the initial stages of cell attachment to glass, steel and aluminium surfaces. It was found that while VSI gave unambiguous results with opaque surfaces, when reflective surfaces were used, an artifact sometimes appeared, with the bacteria appearing as rod-shaped pits rather than as cells on the surface. When the bacteria were altered to increase opacity, this artifact disappeared, and upon further investigation, it was found that the observational artifact was the result of a conflict between light reflected from the bacteria and the light reflected from the bacteria–metal interface. These results suggest that not only can bacteria be measured on surfaces using VSI, but with some modifications to the analytical software, there may be a unique window for studying the bacterial/substrate interface that can be used for quantitative observations. Imaging and characterization of the bacteria–substrate interface in vivo (previously invisible) will provide new insights into the interactions that occur at this important juncture.