Nis elements c er software

Images of cultured cells were recorded under a confocal microscope (A1; Nikon, Tokyo, Japan). The primary antibody for His-tagged ribosomes was anti-His tag antibody (Abcam, ab18184; 1:200), and the secondary antibody was Cy3-conjugated anti mouse-IgG antibody (Jackson ImmunoResearch, 115-165-166, 1:200).
The following is a list of instrument parts and settings used in this study. Laser lines at 405 and 561 nm were used for excitation of DAPI and Cy3, respectively. An oil-immersion objective lens (Apo TIRF 100× Oil, NA = 1.49; Nikon) was used to capture high-magnification images. The pixel size of the confocal images was set to 30 nm. The pinhole size was set to 0.3–0.6 AU. To enhance the resolution, confocal images were deconvolved by using NIS-Elements C-ER software (Nikon) in some cases. All images were recorded at RT (25.5 °C ± 0.5 °C).

The bacterial cells, which were cultured until the late logarithmic phase, were dropped onto a microscope slide and covered with a coverslip. Then, the cell samples were observed, and images were acquired using a confocal microscope (Nikon C2 plus, Yokohama, Japan). The excitation beam for mKO2/mCherry/DsRed/tdTomato was set at 561 nm and the emission signal for mKO2/DsRed/tdTomato was captured at 560–595 nm, while the λ emission for mCherry was 600–650 nm. As for GFP+/EGFP/Staygold, λ excitation = 488 nm and λ emission = 500–550 nm were used. The image analysis was performed using the NIS-ELEMENTS C-ER software (Nikon, Yokohama, Japan). All the fluorescent images acquired using the confocal microscope were captured and processed using identical parameters.

The slides were examined on a Leica DMi8 fluorescent inverted microscope with an HC PL APO 100×/1.40–0.70 oil objective using Leica Application Suite X (LAS X) software v.4.70 (Leica Microsystems, Wetzlar, Germany). Confocal imaging was done using a Nikon A1R Confocal Laser Scanning Inverted Microscope with a Plan Apo 63×/1.4 oil objective using NIS Elements C-ER software v.4.50 (Nikon Instruments Inc., Mel-ville, NY, USA). Images of z-stacks of 15–20 slices × 0.4 µm per specimen were acquired. The lasers and filters used were as follows: 406 nm, filter 450/50 for DAPI; 492 nm, filter 525/50 for AF488; 561 nm, filter 595/50 for Cy3; and 639 nm, filter 700/75 for AF647.
For three-dimensional (3D) reconstruction modeling and analysis, images of interphase nuclei with γH2AX, RNAPII, and S9.6 foci were processed using the Imaris Surface reconstruction module and the Spots Module (Bitplane). RAD51, LigIV, RNAPII, and S9.6 foci on chromosomes were counted manually.