Biofilm thickness measurements by light microscopy

Abstract Light microscopy has been used to measure biofilm thickness. The vertical displacement of the sample required to focus from the biofilm-liquid interface to the biofilm-substratum interface is measured by the stage micrometer. Biofilm thickness is proportional, but not equal, to the measured vertical displacement. An expression for the proportionality constant, kf, in terms of refractive indices is determined from a geometric analysis of the light path. kf can be estimated as the ratio of the refractive index of the film to the refractive index of the media interfacing the film between the objective lens and the sample. The thickness of any transparent film may be determined by light microscopy when the refractive index of the film is known.