Development and validation of the Azu-Eos as a fluorescence-free staining method for enhanced bright-field microscopy...

The comet assay is a valuable technique for assessing cellular DNA damage due to intrinsic and environmental factors, particularly in studies of genotoxicity and mutagenesis. However, its broader application is limited by the need for fluorescent staining, the requirement for expensive fluorescent microscopes, and the inability to preserve and re-examine slides over time. We developed the Azure B-Eosin Y (Azu-Eos) staining method to visualise the comets under a standard bright-field microscope to overcome these limitations. The newly developed DNA-staining technique eliminates the need for fluorescence and allows unlimited slide storage and re-examination without quality deterioration. We optimised various factors to ensure the reliability and quality of bright-field stained comets, achieving results comparable to and even better than those obtained with traditional fluorescent dyes. This cost-effective, sensitive, and fluorescence-free method has broad potential applications in medicine, ecology, and environmental monitoring, enabling fast and on-site genotoxicity testing.