Biofilms are communities of microorganisms that grow on surfaces. Some play important roles in natural processes, but others—such as the plaque on our teeth or the buildup on a hull that reduces the ship’s fuel efficiency—can cause problems. Whether the surface is natural or manufactured, the key requirement for biofilms is that the surface is wet. That, inherently, is what makes them difficult to measure.

To study biofilms, researchers need to know their structures—how thick they are and how "rough" they make the surface. For some of the common 3-dimensional imaging tools, the wet samples must be killed and dried before measurement. Researchers however want to examine biofilms in a non-destructive way, so they can observe changes over time. One such technique, called white light interferometry, has promising vertical resolution and a large field of view, but researchers have rarely used it to measure the soft surfaces of living organisms. This optical microscope depends on reflected light, and because biofilms are mostly water, the biofilm’s surface does not reflect enough light to resolve it from its surrounding liquid environment.

To solve this issue, a PNNL team developed an approach to measure biofilms using white light interferometry. The team discovered that adding a tiny air bubble between the biofilm’s surface and a cover slip is the essential trick; it makes the underlying biofilm structure highly reflective and therefore measurable. The method works for different types of apparatuses, from ones using flow cells to others using inverted multi-well plates.

The ability to monitor the "roughness" of a surface is an advantage. The researchers found that surface roughness increases sooner than surface thickness, providing an early indicator of biofilm growth. In addition, because the biofilm can be monitored continuously over time, scientists can use the imaging tool to observe how the biofilm responds to a changing environment, such as introduced chemicals or drugs. The new method provides a window into how to treat harmful biofilms.