Fig. 9: Schematic overview of creating computational microscope images using PyFoci. | Communications Biology

Fig. 9: Schematic overview of creating computational microscope images using PyFoci.

From: A computational approach to quantifying miscounting of radiation-induced double-strand break immunofluorescent foci

Fig. 9

The generation of simulated DNA DSBs and repair (steps 1–2) is independent of PyFoci and can be interchanged with other models. Any model which can output DSB position as a function of time can use PyFoci to create computational microscope fluorescent foci images (for at least the Ku/DNA-PKcs marker visualisation). Steps 3a and 3b represent the choice in the visualisation approach, whilst Ku/DNA-PKcs marker only requires information on the DSB spatial distribution, the γ-H2AX marker requires information on the chromosome geometry, namely the topologically associated domains the DSBs are created in. Steps 4–7 are the same regardless of the visualisation marker, the only difference is what biological object is being fluoresced (DSBs or γ-H2AX). Step 5 requires the microscopes PSF to be defined for the desired microscope and magnification under evaluation. Step 6 requires the user to define at what time point they wish to create the microscope image and at what z-axis slice. Step 7 creates the image and performs the foci counting on the produced image, this can be done with the in-built foci counter or can be exported to external foci counting software.

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