Objective threshold selection procedure (OTS) for segmentation of scanning laser confocal microscope images.

The determination of volumes and interface areas from confocal laser scanning microscopy (CLSM) images requires the identification of component objects by segmentation. An automated method for the determination of segmentation thresholds for CLSM imaging of biofilms was developed. The procedure, named objective threshold selection (OTS), is a three-dimensional development of the approach introduced by the popular robust automatic threshold selection (RATS) method. OTS is based on the statistical properties of local gray-values and gradients in the image. By characterizing the dependence between a volumetric feature and the intensity threshold used for image segmentation, the former can be determined with an arbitrary confidence level, with no need for user intervention. The identification of an objective segmentation procedure renders the possibility for the full automation of volume and interfacial area measurement. Images from two distinct biofilm systems, acquired using different experimental techniques and instrumental setups were segmented by OTS to determine biofilm volume and interfacial area. The reliability of measurements for each case was analyzed to identify optimal procedure for image acquisition. The automated OTS method was shown to reproduce values obtained manually by an experienced operator.

[1]  Z Lewandowski,et al.  Oscillation characteristics of biofilm streamers in turbulent flowing water as related to drag and pressure drop. , 1998, Biotechnology and bioengineering.

[2]  M. V. van Loosdrecht,et al.  Heterogeneity of biofilms in rotating annular reactors: Occurrence, structure, and consequences , 1994, Biotechnology and bioengineering.

[3]  A. Jayaraman,et al.  Characterization of axenic Pseudomonas fragi and Escherichia coli biofilms that inhibit corrosion of SAE 1018 steel , 1998, Journal of applied microbiology.

[4]  Bjarke Bak Christensen,et al.  In Situ Gene Expression in Mixed-Culture Biofilms: Evidence of Metabolic Interactions between Community Members , 1998, Applied and Environmental Microbiology.

[5]  A. Bull,et al.  4-Chlorophenol degradation by a bacterial consortium: development of a granular activated carbon biofilm reactor , 1999, Applied Microbiology and Biotechnology.

[6]  Zbigniew Lewandowski,et al.  Effects of biofilm structures on oxygen distribution and mass transport , 1994, Biotechnology and bioengineering.

[7]  Gary J. Harkin,et al.  Quantifying biofilm structure using image analysis. , 2000, Journal of microbiological methods.

[8]  J. Wimpenny,et al.  A unifying hypothesis for the structure of microbial biofilms based on cellular automaton models , 1997 .

[9]  B. Christensen,et al.  Establishment of New Genetic Traits in a Microbial Biofilm Community , 1998, Applied and Environmental Microbiology.

[10]  J J Heijnen,et al.  Mathematical modeling of biofilm structure with a hybrid differential-discrete cellular automaton approach. , 1998, Biotechnology and bioengineering.

[11]  Z Lewandowski,et al.  Biofilms, the customized microniche , 1994, Journal of bacteriology.

[12]  Jones,et al.  Surface‐catalysed disinfection of thick Pseudomonas aeruginosa biofilms , 1998, Journal of applied microbiology.

[13]  G. Booth,et al.  BacSim, a simulator for individual-based modelling of bacterial colony growth. , 1998, Microbiology.

[14]  P. Wilderer,et al.  Fractal structure of biofilms: new tools for investigation of morphology , 1995 .

[15]  B. Christensen,et al.  Distribution of Bacterial Growth Activity in Flow-Chamber Biofilms , 1999, Applied and Environmental Microbiology.

[16]  Michael H. F. Wilkinson,et al.  Digital Image Analysis of Microbes: Imaging, Morphometry, Fluorometry, and Motility Techniques and Applications , 1998 .

[17]  J. Lawrence,et al.  Determination of Diffusion Coefficients in Biofilms by Confocal Laser Microscopy , 1994, Applied and environmental microbiology.

[18]  Josef Kittler,et al.  Threshold selection based on a simple image statistic , 1985, Comput. Vis. Graph. Image Process..

[19]  P A Wilderer,et al.  Automated Confocal Laser Scanning Microscopy and Semiautomated Image Processing for Analysis of Biofilms , 1998, Applied and Environmental Microbiology.

[20]  J. Costerton,et al.  Optical sectioning of microbial biofilms , 1991, Journal of bacteriology.