Imaging nucleic acids in biofilms.

Extracellular nucleic acids (eNA) are ubiquitous components of bacterial biofilms, contributing to structural integrity, antimicrobial resistance, and immune evasion. While traditionally viewed as extracellular DNA in the canonical B-form, recent discoveries reveal remarkable structural diversity within biofilm matrices, including left-handed Z-DNA, G-quadruplexes, i-motifs, triplexes, and extracellular RNA. These non-canonical structures possess emergent properties distinct from B-DNA: they resist degradation by mammalian DNase I, they form catalytically active DNAzymes with peroxidase activity, and they serve as conduits for extracellular electron transfer. Despite their biological significance, these structures have been largely overlooked due to limitations in visualisation methods. The present review critically evaluates current tools for imaging eNA in biofilms, with emphasis on fluorescent DNA-binding dyes and structure-specific immunolabelling approaches. The widely used DNA-binding dyes, such as propidium iodide and TOTO™-1, exhibit strong structural biases and render non-canonical structures invisible. This has important implications for interpreting nuclease treatment outcomes and biofilm composition. We provide practical guidance for comprehensive eNA visualisation, recommending integration of multiple detection methods: combining far-red dyes with TOTO™-1 for broad structural coverage and validating findings with commercial monoclonal antibodies (Z22, BG4, 1H6, iMab, Jel466, and J2) alongside functional assays. Understanding eNA structural diversity and developing appropriate detection tools are essential for rational design of structure-specific nuclease therapeutics targeting biofilm infections.