Du 897 camera

We used a dSTORM system, which allows imaging at approximately 20 nm resolution by using photo-switchable fluorophores (all dSTORM imaging was done on TIRF mode). Five μm brain slices were mounted on poly-D-lysine coated coverslips (no. 1.5 H, Marienfeld-superior, Lauda-Königshofen, Germany). dSTORM imaging was performed in a freshly prepared imaging buffer containing 50 mM Tris (pH 8.0), 10 mM NaCl and 10% (w/v) glucose with an oxygen-scavenging GLOX solution (0.5 mg/ml glucose oxidase (Sigma-Aldrich)), 40 μg/ml catalase (Sigma-Aldrich), 10 mM cysteamine MEA (Sigma-Aldrich), and 1% β mercaptoethanol (Barna et al., 2016 (link); Dempsey et al., 2011 (link); Zhang et al., 2016 (link)). A Nikon Ti-E inverted microscope was used. The N-STORM Nikon system was built on TIRF illumination using a 1.49 NA X100 oil immersion objective and an ANDOR DU-897 camera. 488, 568 and 647 nm laser lines were used for activation with cycle repeat of ~8000 cycles for each channel. Nikon NIS Element software was used for acquisition and analysis; analysis was also performed by ThunderSTORM (NIH ImageJ [Ovesný et al., 2014 (link)]). Images in 2D were Gaussian fit of each localization; in the N-STORM software.

For PALM a Nikon Ti microscope (N-STORM system) with a SR Apo TIRF 100x objective was used. TIRF laser position of motorized TIRF illumination unit was set to about 4200 μ▯ and finely tuned to achieve highest contrast at the level of 1 μm above the coverslip surface^{46 (link)}. For photoconversion/excitation 405 nm laser (5% intensity) and 561 nm laser (100% intensity) were used 43. Images were recorded with an Andor DU-897 camera operating in copped mode (256 × 256 pixels with 160 nm effective pixel size). Exposure time was 30 ms/frame. The system operated in sequential mode using excitation with a 405 nm laser for 1 frame and then excitation with a 561 nm laser for 3 frames (1 – registration of 561 nm signal, 2,3 – bleaching of the 561 nm signal), 500 cycles overall. Every second frame of a cycle was used for analysis. Images were analyzed using the ThunderSTORM plugin of ImageJ^{47 (link)}.

A Nikon Ti-E inverted fluorescence microscope with a 100X NA 1.40 oil immersion phase contrast objective was used for imaging the bacteria. Fluorescence was excited by a 200W Hg lamp through an ND4 or ND8 neutral density filter. Chroma 41004, 41001 and 31000v2 filtercubes were used to record mCherry, GFP and DAPI images, respectively. Images were captured by an Andor iXon DU897 camera and recorded using NIS-Elements software.
Cells were imaged on M9 agar pads for still imaging. For time lapse imaging home-made glass bottom dishes were used. Cells were pipetted to #1.5 glass coverslips on the bottom of the dish and covered with about 1 cm thick slab of M9 agar. No antibiotics were used in M9 agar during imaging. Agar was supplemented with IPTG (10–40 µM) for strains with ZipA-GFP constructs. For DAPI labeling cells were incubated in 0.2 µg/ml DAPI for 1/2 hour before spreading cells on the pads.

Cells were prepared and immobilized on 1.2% agarose slides as described above. To reduce binding of Nile Red to the coverslip surface, which can suppress the structured illumination pattern, the coverslips were coated with l-dopamine as described before (28 (link)). In brief, l-dopamine (2 mg/mL freshly dissolved in 1 mM Tris pH 8.0) was added to the coverslip and incubated at room temperature for 30 min. The excess l-dopamine and Tris were then removed by aspiration and submersion in H₂O followed by evaporation at 37°C for 30 min. Dual-color two-dimensional (2D)-SIM was performed using Nikon N-SIM equipped with 488- and 561-nm lasers, Nikon CFI SR HP Apochromat TIRF 100×/1.49 oil objective, and Andor iXon DU-897 camera. Image capture and SIM reconstruction was carried out with NIS Elements 5.21 (Nikon).