Plan fluotar objective

Effects of the treatments were directly analysed on the microscopic slides under the microscope (about 500–2000 cells) and/or on acquired 3D images (>50 cells) (below). Two replicas of microscopic slides for each sample were directly evaluated by two experienced scientists. The following equipment was used for confocal microscopy and image acquisition: an automated Leica DM RXA fluorescence microscope (Leica, Wetzlar, Germany) equipped with an oil immersion Plan Fluotar objective (100×/NA1.3) and a CSU 10a Nipkow disc (Yokogawa, Japan); a CoolSnap HQ CCD-camera (Photometrix, Tuscon, AZ, USA); and an Ar/Kr-laser (Innova 70 C Spectrum, Coherent, Santa Clara, CA, USA)^{23 (link)}. Automated exposure, image quality control and other procedures were performed using Acquarium software^{24 (link)}. The exposure time and the dynamic range of the camera in the red, green and blue channels (R-G-B) were adjusted to the same values for all slides to obtain quantitatively comparable images. Forty serial optical sections were captured at 0.3-μm intervals along the z-axis.

The immunofluorescence of the detected proteins was analyzed using images obtained with a high-resolution Leica DM RXA confocal microscope (Leica, Wetzlar, Germany) equipped with an oil immersion Plan Fluotar objective (100×/NA1.3) and a CSU 10a Nipkow disk (Yokogawa, Japan) for confocal imaging. A CoolSnap HQ CCD-camera (Photometrix, Tuscon, AZ, USA) and an Ar/Kr laser (Innova 70C Spectrum, Coherent, Santa Clara, CA, USA) were used for image acquisition. Automated exposure, image quality control, image analysis, and other procedures were performed using Acquiarium software [30 (link)]. The exposure time and dynamic range of the camera in the red, green, and blue channels were adjusted to the same values for all slides to obtain quantitatively comparable images. Forty serial optical sections were captured at 0.2 mm intervals (along the z-axis). In total, 100–300 cells were recorded for each set of conditions, and the experiments were repeated three times. The results are reported as standard error of mean. A t-test was used for the statistical comparison of specified effects obtained in cell nuclei after γ-irradiation and before irradiation.