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

Abstract

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.

Critical analysis of methods to determine growth, control and analysis of biofilms for potential non-submerged antibiofilm surfaces and coatings

“…Microscopy is widely used in the articles described in this review, and indeed across the biofilm literature. There are many different approaches that can be used to quantify biofilm by microscopy including bespoke software options [ 100 , 125 ], but this review demonstrates that often researchers may opt to using images where biofilm is absent in lieu of quantitative data. Whilst this type of image is of value, due to the nature of microscopy only showing a very small portion of a surface, it is important that researchers provide robust methodology to describe how much of a surface they analysed, which is critical to understanding the scale of antimicrobial effect.…”

A Comprehensive Review of Recent Research into the Effects of Antimicrobial Peptides on Biofilms—January 2020 to September 2023

“…Biofilms comprise an extracellular matrix (ECM), containing a range of extracellular polymeric substances (EPS), including cellular debris, proteins, polysaccharides, lipids, and nucleic acids [3], and the formation of biofilms relies on key signaling molecules such as c-di-GMP, proteins, and small regulatory RNAs (sRNAs), as well as quorum sensing (QS) [4]. Microscopic and biochemical techniques help in morphological analysis and comprehending the formation and variability of biofilms [5].…”