The following customizations were most favored by our clients:

• Standardized assessment of foci in cell nuclei using the Metafer object detection capabilities.
• Automated Z-stack imaging.
• Automated exposure control based on image quality.
• Help to detect co-localizations.
• Standardization of foci counts due to background condition and foci size settings.

Double-strand breaks (DSB) are the most deleterious DNA lesions that, if left unrepaired, may have severe consequences for cell survival. They are known to lead to chromosome aberrations, genomic instability, and cell death. Various physical, chemical, and biological factors are involved in DSB induction. The histone H2AX, a protein contributing to the nucleosome-formation and therefore the structure of DNA, becomes phosphorylated on serine 139, then called γ-H2AX, as a reaction to the formation of DSBs. Since γ -H2AX expands a large distance from the DSB site, and since specific γ-H2AX antibodies are available, the DSBs can be visualized as discrete foci. Quantification of γ-H2AX foci can be used to detect the genotoxic effect of different toxic substances, including ionizing radiation.

Since γ-H2AX foci appear as spot-like signals under the microscope, Metafer users can configure a workflow that utilizes Metafer's spot-counting features to automatically generate foci-count proposals, which can then be reviewed by the user. Unlike FISH spots, γ-H2AX foci often vary in size and may merge into larger fluorescent patches in cells exposed to higher doses of DSB-inducing agents. To address this, our application specialists have created routines for Metafer users to automatically count foci in cell nuclei, providing the option to include a weighing factor in the measurement that facilitates the customization of scoring criteria to meet specific standards. These criteria can be fine-tuned using data from validation experiments and established dose-response curves.