Objective threshold selection procedure (OTS) for segmentation of scanninglaser confocal microscope images

The determination of volumes and interface areas from confocal laser scanni ng microscopy (CLSM) images requires the identification of component object s by segmentation. An automated method for the determination of segmentatio n thresholds for CLSM imaging of biofilms was developed. The procedure, nam ed objective threshold selection (OTS), is a three-dimensional development of the approach introduced by the popular robust automatic threshold select ion (RATS) method. OTS is based on the statistical properties of local gray -values and gradients in the image, By characterizing the dependence betwee n a volumetric feature and the intensity threshold used for image segmentat ion, the former can be determined with an arbitrary confidence level, with no need for user intervention. The identification of an objective segmentat ion procedure renders the possibility for the full automation of volume and interfacial area measurement. Images from two distinct biofilm systems, ac quired using different experimental techniques and instrumental setups were segmented by OTS to determine biofilm volume and interfacial area. The rel iability of measurements for each case was analyzed to identify optimal pro cedure for image acquisition. The automated OTS method was shown to reprodu ce values obtained manually by an experienced operator. (C) 2001 Elsevier S cience B.V. All rights reserved.