Deconvolution software

Cell samples were visualized using an Olympus IX71 microscope equipped with a personal Delta Vision system and a Photometrics CoolSnap HQ2 monochrome camera. Stacks of seven Z-series sections were acquired at 0.2-μm intervals. All fluorescence images are maximum two-dimensional projections of Z-series and were analyzed using deconvolution software from Applied Precision (Issaquah, WA). Measurements were made from micrographs using the ImageJ (National Institutes of Health, Bethesda, MD) or MetaMorph (Molecular Devices, Sunnyvale, CA) programs. For calcofluor, cells were harvested (1 ml), washed once, and resuspended in water with 20 μg/ml of calcofluor at room temperature.

Images and cell length measurements were obtained using an Olympus IX71 microscope equipped with a personal Delta Vision system and a Photometrics CoolSnap HQ2 monochrome camera, with Metamorph software (Universal Imaging, Molecular Devices, Downingtown, PA, USA). Measurements were made from micrographs using the IMAGEJ (National Institutes of Health). All microscopy was conducted on live midlog-phase cells placed on slides, except for cultures for DAPI and aniline blue staining, which were fixed in 70% ethanol at room temperature, washed, and pelleted before resuspension in 5 μl of DAPI solution (500 μg/ml). More than 200 dividing cells per strain were measured for the cell length data. Stacks of ten z-series sections were acquired at 0.2-μm intervals. All fluorescence images are maximum two-dimensional (2D) projections of z-series and were analyzed using deconvolution software from Applied Precision. To calculate the area of the nucleus occupied by Rad52p-YFP a binary mask was created using the mean value of the nuclear background without foci as a threshold and measuring the area of this mask. To detect Rad51p indirect immunofluorescence microscopy was performed according to the protocol described in (46 (link)). The rabbit polyclonal anti-human Rad51p (PA5-27195, Thermo Fisher, Rockford, IL, USA) was diluted 1.100.