Em ccd camera

Mice were subjected to sciatic nerve transection and exposure of the ipsilateral gastrocnemius muscle under anesthesia with isoflurane; inhaled anesthesia was maintained throughout the subsequent intravital imaging experiment. Mouse body temperature was maintained using a heating pad. Gastrocnemius muscle intravital imaging experiments were performed using a spinning-disk confocal microscope (Zeiss Axio Observer Z1 basic stand inverted scope equipped with a Yokogawa CSU-X spinning disk unit and an EMCCD camera [Hamamatsu]). Samples were excited with 488-nm and 633-nm laser lines, and emission was collected using a 10×/0.3 and 25×/0.8 objective with standard emission filter sets (Semrock). Images were acquired using ZEN acquisition software (Zeiss). The muscle microvasculature was visualized by intravenous infusion of 5 µg of anti-PECAM-1. Neutrophils inside the muscle microvasculature and tissue were visualized via topical application of fluorescently labeled anti-Ly6G (1A8) antibody.

For single-molecule microscopy, a Nikon Eclipse Ti with a TIRF objective (× 100, Apo, NA: 1.49) was used. Image acquisition was accomplished using a back-illuminated EMCCD camera (Hamamatsu). The center of a 20-fold expanded beam from a 100-mW multiline argon laser (JDS Uniphase, laser head: 2219-G5MLS) was focused on the back focal plane and operated during image acquisition with 150 to 200 W/cm². A 514-nm laser diode was used as excitation source. For image acquisition, the program Andor Solis 4.21 was applied. Streams of 1500 frames of 20 ms were acquired. Cells continued to grow after imaging, showing that there is little to no photo damage during acquisition. Acquired streams were loaded into Fiji ImageJ [75 (link)], and pixel sizes (106 nm) and time increments were calibrated. Tracking of single molecules was achieved using u-track 2.0 [76 (link)]. Only trajectories with at least four frames were used for further analysis to calculate the diffusion coefficients. Trajectory x/y coordinates and diffusion coefficients were calculated using a custom-made MATLAB script which included localization error. At least 1000 tracks were used for each analysis so that the CDF curves had at least 10⁴ points.

Cells were imaged on a Zeiss LSM 700 confocal microscope with Zeiss ×40 and ×63 oil objectives (NA 1.3) with the appropriate filter sets and a back-thinned Hamamatsu EMCCD camera.

We 3D-printed electrotaxis chambers (Figure 2—figure supplement 1) with dimensions of 20 mm × 5 mm × 0.25 mm and composed of a clear resin using a Formlabs Form2 3D-printer. Agar bridges were used to isolate cell media from electrodes to minimize electrochemical products and pH changes. Twenty V/cm constant EFs were applied. Time-lapse images of the phase-contrast channel and the RFP/GFP channel were recorded using PerkinElmer spinning-disk microscope at a frame rate of 0.1 frames/s (Yokogawa CSU-X1 spinning-disk scan head (5000 rpm)) with Hamamatsu EMCCD camera and Volocity analysis software.

Escherichia coli cells (Stellar) carrying pJYB213 (ParBF‐eGfp), grown in mid‐exponential phase in MGC medium and spotted on microscope slides coated with 1% MGC‐buffered agarose, were subjected to laser‐bleaching. Each field was imaged three times (pre‐bleached step) before photobleaching (at ~2.4 s) a single ParB_F focus with a 488 nm laser into two‐foci cells. ROI (region of interest) were 0.2 × 0.2 or 0.3 × 0.3 corresponding to 5 or 9 pixels, respectively. The laser power was set between 67 and 74 Hz to ensure partial bleaching, thus enabling to follow fluorescence recovery on a time scale of second right after photobleaching. Images were taken using an EMCCD camera with a 0.13 μm per pixel resolution (Hamamatsu). To follow recovery dynamics, images were taken every 5, 10, and 20 s up to 38, 108, and 169 s, respectively. Overall, photobleaching in the field of view during the time course of each FRAP experiment (green curves in Fig 6B and Appendix Fig S6A) was averaged from 15 unbleached foci from each field. Normalization to 1 was performed by averaging the focus fluorescence intensity from the three pre‐bleached images.