Imagem x2 em ccd

For this experiment, the streptococcal SpeB protocol for Streptococcus by Rosch and Caparon [75 (link)] was adapted to Bacillus. Strains were cultivated in LB medium at 37°C and 200 rpm with the addition of 25 µg/ml kanamycin until the transitional growth phase. The culture was pelleted at 4000 rpm for 2 min and resuspended in fresh LB containing 1% of the “DQ starch substrate stock solution” (1 mg/ml, EnzChek Ultra Amylase Assay Kit, Invitrogen Detection Technologies, Carlsbad, CA, USA). Cells were mounted on ultrapure-agarose slides dissolved in LB (1%) for immobilization of cells and incubated for 30 min at 37°C.
Imaging was performed via epi-fluorescence microscopy, using a Nikon Eclipse Ti-E, Nikon Instruments Inc with a CFI Apochromat objective (TIRF 100 × oil, NA 1.49) and an EMCCD camera (ImagEM X2 EM-CCD, Hamamatsu Photonics KK). The samples were illuminated with Nikon C-HGFIE Intensilight (Precentered Fiber Illuminator) and the YFP-channel filter cube ET 500/20, T 515 LP, ET 535/30. Images were processed with MetaMorph (version 2.76), and ImageJ [45 (link)].

A total of 5 µL of the culture was placed onto a clean glass coverslip and topped with an agar pad containing the growth medium. For the agar pad, two smaller slides were used. A total of 100 µL water with 1% agar was placed between the two slides, and after cooling, one of the two slides was removed, and the agar was placed on the sample. Single molecule tracking of the cultures was performed with a customized slim-field setup. The microscope was a Nikon Eclipse Ti-E (Nikon Instruments Inc., Melville, NY, USA), an inverted fluorescence microscope. The camera used was an ImagEM X2 EM-CCD (Hamamatsu Photonics KK, Shizuoka, Japan). The laser of the setup was the center of a 514 nm laser diode (max power 100 mW, TOPTICA BEAM Smart, Pittsfield, MA, USA). The intensity was 20% (about 160 W cm⁻² in the image plane). A CFI Apochromat objective (TIRF 100 x Oil, NA 1.49) was used. The videos of the samples have 3000 frames. The movies of mNeongreen fusions were cut after 1000 frames, the mVenus fusions were cut after 500 frames, in order to reach single molecule level. After cutting the movies, the cell meshes were set with Oufti [18 (link)], and the trajectories’ analysis was performed with Utrack [19 (link)]. The final analysis was performed by SMTracker [20 (link),21 (link)]. General data for movies see Table S2.