Coolsnap hq2 camera

S. pneumoniae strains were grown until early exponential phase and observed by fluorescence microscopy on a thin layer of 1% (m/v) agarose in PreC medium [51 (link)]. Images were acquired in a Zeiss Axio Observer microscope, equipped with a Photometrics CoolSNAP HQ2 camera (Roper Scientific), with appropriate exposure times (500–1000 ms for GFP; 5000 ms for Citrine and CFP; 100 ms for AlexaFluor secondary antibody), and analyzed using FIJI software [57 (link)] as well as eHooke software [58 (link)], which was developed in-house and is available at https://github.com/BacterialCellBiologyLab/eHooke.
Determination of the fluorescence ratio (FR) was performed as previously described [35 (link)]. Briefly, the intensity of the fluorescent signal at the division septum was divided by the fluorescent signal at the peripheral membrane. Average background fluorescence was subtracted from every value. An FR value higher than 2 is indicative of septal enrichment. Quantification was performed for at least 100 cells of each strain.
In vivo detection of the capsule produced at the surface of S. pneumoniae cells was performed as previously described [28 (link)], but Anti-rabbit Alexa Fluor 594 antibody (Invitrogen) was used as a secondary antibody. When necessary LytA-GFP purified protein was added to the media at 5 μg/ml concentration and incubated for 10 min at 37°C.

R-loop immunostaining and fluorescence quantification was performed as described [116 (link)], with the exception of cell lysis, where we mechanically broke cells in liquid nitrogen instead of using enzymatic lysis. R-loops were visualized using ⍺S9.6 antibody [117 (link)]. For negative control samples, we added RNase H (Roche 10786357001) at 3u/100μl and incubated for two hours at 37°C. Nuclei were stained with DAPI at 3μg/mL in 50% glycerol. Images were obtained using a spinning disk confocal microscope (Yokogawa CSU-X1 head mounted on Olympus body), CoolSnap HQ2 camera (Roper Scientific), and a 100X Plan Apochromat 1.4 NA objective (Olympus). Images presented correspond to maximal projections of 10 slides’ stacks using Image J open software [118 (link)].

Spinning-disk confocal time-lapse imaging was done at 37°C in a thermostat-controlled chamber using an Eclipse 80i microscope (Nikon) equipped with spinning disk confocal head (Perkin), a 100× objective, and either an Ultra897 iXon camera (Andor) or CoolSnapHQ2 camera (Roper Scientific). Fixed samples were imaged using a 60× objective with the same setup. TIRF microscopy was done using Leibovitz’s medium (Life Technologies) at 37°C in a thermostat-controlled chamber. An Eclipse Ti inverted microscope (Nikon) equipped with either a TIRF module (Nikon) or an iLAS2 azimuthal TIRF module (Roper Scientific), a 100× TIRF objective, a beam splitter (Roper Scientific), and an Evolve 512 electron-multiplying CCD camera (Photometrics) was used in this case (Boulanger et al., 2014 (link)). All acquisitions were driven by Metamorph (Molecular Devices).

For in vivo imaging, cell cultures were grown to exponential phase. Cells were pelleted by centrifugation at 3000 rpm and resuspended in the residual media. Cells were placed on microslide wells (Ibidi, # 80,821) coated with soybean lectin (Sigma, #L1395). Images were acquired using a Spinning-disk confocal microscope (IX-81; Olympus; CoolSnap HQ2 camera, Plan Apochromat 100x-1.4 NA objective [Roper Scientific]). MetaMorph software was used for image acquisition. Temperature was stably controlled at 25 °C during imaging. For fixed cells, a DAPI-calcofluor staining was used. Cells fixed with 70% ethanol were washed with PBS and pellet was resuspended in 5 μl of the DAPI/calcofluor mounting solution (100 μl Mounting solution contains: 38 μl glycerol 50%, 46.5 μl H₂O, 10 μl Antifade (p-phenylenediamine, Sigma #P6001; 10 mg/ml in phosphate-buffered saline pH 8.2), 3 μl DAPI (4, 6-diamidino-2-phenylindole, Sigma #D1388; 0.1 mg/ml), and 2.5 μl calcofluor white (Sigma #F6259; 0.35 mg/ml in H₂O).