AUTOMATED CONFOCAL LASER-SCANNING MICROSCOPY AND SEMIAUTOMATED IMAGE-PROCESSING FOR ANALYSIS OF BIOFILMS

The purpose of this study was to develop and apply a quantitative opti cal method suitable for routine measurements of biofilm structures und er in situ conditions. A computer program was designed to perform auto mated investigations of biofilms by using image acquisition and image analysis techniques. To obtain a representative profile of a growing b iofilm, a nondestructive procedure was created to study and quantify u ndisturbed microbial populations within the physical environment of a glass flow cell. Key components of the computer-controlled processing described in this paper are the on-line collection of confocal two-dim ensional (2D) cross-sectional images from a preset 3D domain of intere st followed by the off-line analysis of these 2D images. With the quan titative extraction of information contained in each image, a three-di mensional reconstruction of the principal biological events can be ach ieved. The program is convenient to handle and was generated to determ ine biovolumes and thus facilitate the examination of dynamic processe s within biofilms. In the present study, Pseudomonas fluorescens or a green fluorescent protein-expressing Escherichia coli strain, EC12, wa s inoculated into glass flow cells and the respective monoculture biof ilms were analyzed in three dimensions. In this paper we describe a me thod for the routine measurements of biofilms by using automated image acquisition and semiautomated image analysis.