title: A two-step procedure for automatic and accurate segmentation of volumetric CLSM biofilm images.
abstract: This paper presents a robust two-step segmentation procedure for the study of biofilm structure. Without user intervention, the procedure segments volumetric biofilm images generated by a confocal laser scanning microscopy (CLSM). This automated procedure implements an anisotropic diffusion filter as a preprocessing step and a 3D extension of the Otsu method for thresholding. Applying the anisotropic diffusion filter to even low-contrast CLSM images significantly improves the segmentation obtained with the 3D Otsu method. A comparison of the results for several CLSM data sets demonstrated that the accuracy of this procedure, unlike that of the objective threshold selection algorithm (OTS), is not affected by biofilm coverage levels and thus fills an important gap in developing a robust and objective segmenting procedure. The effectiveness of the present segmentation procedure is shown for CLSM images containing different bacterial strains. The image saturation handling capability of this procedure relaxes the constraints on user-selected gain and intensity settings of a CLSM. Therefore, this two-step procedure provides an automatic and accurate segmentation of biofilms that is independent of biofilm coverage levels and, in turn, lays a solid foundation for achieving objective analysis of biofilm structural parameters.
authors.affiliation: Electrical and Computer Engineering, Schulich School of Engineering, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4.
authors.forename: Jerome
authors.initials: J
authors.lastname: Yerly
authors.forename: Yaoping
authors.initials: Y
authors.lastname: Hu
authors.forename: Steven M
authors.initials: SM
authors.lastname: Jones
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Martinuzzi
mesh_terms: D016880:Anisotropy; D018441:Biofilms / Q000254:growth & development*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D018613:Microscopy, Confocal / Q000295:instrumentation / Q000379:methods*; D011513:Proteus mirabilis / Q000502:physiology; D013211:Staphylococcus aureus / Q000502:physiology