Na oil immersion objective

Cell or cell lysate samples were placed on a glass dish (NEST Biotechnology, USA) and covered with agar before micrographs were acquired at 37 °C (for the re-culturing cell samples) or 30 °C (for all other samples) with an N-SIM imaging system (Nikon, Japan) using the 2D-SIM mode, a ×100/1.49 NA oil-immersion objective (Nikon, Japan), and under excitation of a 488 or 561 nm laser beam. The 3D images were acquired with an N-SIM imaging system using the 3D mode. The samples were sectioned every 120 nm along the Z-axis. The images were further reconstructed using the NIS-Elements AR 4.20.00 (Nikon, Japan) before a further processing with the GNU image manipulation program. DV Elite microscope with specific optics was also utilized to perform live-cell imaging analysis. At least four images were obtained, and more than 50 bacterial cells were examined for each experiment. All experiments were biologically repeated at least three times.

The fixed samples were imaged on an automated inverted Nikon Ti-2 wide-field microscope equipped with ×60 1.4NA oil immersion objective lens (Nikon), Spectra X LED light engine (Lumencor), and Orca 4.0 v2 scMOS camera (Hamamatsu). The live cell experiments were performed on a custom microscope built around Nikon Ti-E stand. The excitation was through HTIRF (Nikon) with an LU-n4 four laser unit (Nikon) with solid state lasers with wavelengths 405, 488, 561, and 640 nm. The main dichroic was a quad band dichroic mirror (Chroma, ET-405/488/561/640 nm laser quad band set for TIRF applications). The imaging was done through the ×100 1.49NA oil immersion objective (Nikon). To achieve simultaneous 3-color imaging, we used a TriCam light splitter into three separate EMCCD cameras (Andor iXon Ultra 897) with ultraflat 2 mm thick imaging splitting dichroic mirrors (T565LPXR-UF2, T640LPXR-UF2). A band pass emission filter was placed in front of each camera, respectively (ET525/50 m, ET595/50 m, and ET655lp). The microscope was also equipped with an automated XY-stage with extra fine lead-screw pitch of 0.635 mm and 10 nm linear encoder resolution and a Piezo-Z stage (Applied Scientific Instrumentation) for fast Z-acquisition. The whole microscope was under the control of Nikon Elements for automation.