Tools of the Trade: Image Analysis Programs for Confocal Laser-Scanning Microscopy Studies of Biofilms and Considerations for Their Use by Experimental Researchers

[Image: see text] Confocal laser-scanning microscopy (CLSM) is the bedrock of the microscopic visualization of biofilms. Previous applications of CLSM in biofilm studies have largely focused on observations of bacterial or fungal elements of biofilms, often seen as aggregates or mats of cells. However, the field of biofilm research is moving beyond qualitative observations alone, toward the quantitative analysis of the structural and functional features of biofilms, across clinical, environmental, and laboratory conditions. In recent times, several image analysis programs have been developed to extract and quantify biofilm properties from confocal micrographs. These tools not only vary in their scope and relevance to the specific biofilm features under study but also with respect to the user interface, compatibility with operating systems, and raw image requirements. Understanding these considerations is important when selecting tools for quantitative biofilm analysis, including at the initial experimental stages of image acquisition. In this review, we provide an overview of image analysis programs for confocal micrographs of biofilms, with a focus on tool selection and image acquisition parameters that are relevant for experimental researchers to ensure reliability and compatibility with downstream image processing.