Elucidating bacteriophage dynamics and interactions with real-time label-free optical imaging

Abstract Bacteriophages are the focus of extensive research, and monitoring their dynamics and interactions with bacterial hosts is crucial to characterize the mechanisms of infection and to support potential applications in biotechnology and medicine. Traditional monitoring techniques rely on the fluorescent labelling of bacteriophages due to their size being nanometric. In this paper, we propose a novel, label-free method to generate optical signatures of bacteriophages in a conventional microscopy set-up by exploiting the optical phenomenon of caustics. Using Pseudomonas aeruginosa phages (pelp20 and phiKZ) and a newly isolated Escherichia coli phage (EcoLiv25), we demonstrate the generation of distinct optical signatures that enable prolonged monitoring of phage dynamics in liquid media. The results obtained demonstrate caustics-based optical microscopy as a robust, non-invasive approach that complements existing imaging methods by enabling real-time observation and quantitative analysis of phage motion, transport and interactions. Building on these capabilities, the technique can accelerate fundamental studies of viral dynamics and ecology, enable real-time observation of phage–bacteria interactions and support the development of phage-based diagnostics and antimicrobial therapies.