Filter set 10

EPI or TIRF illuminations were used for our experiments. In Bafilomycin A1 experiments, EPI and TIRF illuminations were alternated in real time for whole-cell pHluorin fluorescence (pHF) signal measurements in individual cells, in basal condition and upon stimulation. By imaging with excitation light at 488 nm generated by a 488 nm laser (20 mW; Laserphysics) and by a polychromator illumination system (Visichrome), the pHF signal was recorded at 10 Hz through a 100X objective lens (α-plan FLUAR 100X, 1.45 NA, Zeiss, Germany) and filtered with Zeiss filter set 10 (Zeiss) [19] (link). For TIRF illumination, the expanded beam (488/568 nm argon/krypton multilinelaser, 20 mW) (Laserphysics, USA) was passed through an AOTF laser wavelength selector (VisiTech International) synchronized with a SNAP-HQ CCD camera (Roper Scientific, Germany) under Metafluor software (Universal Imaging, USA) control and was introduced from the high numerical aperture objective lens (α-plan FLUAR 100X, 1.45 NA, Zeiss, Germany). Light entered the coverslips and underwent total internal reflection at the glass-cell interface. In our experimental conditions, penetration depth of TIRF illumination was calculated to be about 90 nm [20] (link). Light was filtered with a beam splitter (Zeiss filter set 10). Images were acquired at 20–40 Hz. Pixel size 126 nm at binning 2.

Overnight cultures of relevant strains in LB or SMS medium were diluted to an optical density at 600 nm (OD₆₀₀) of 0.05 and grown at 37°C in shaking flasks. Samples of 1 to 2 μl were taken in early exponential phase (OD₆₀₀ of 0.2 to 0.4) or in early stationary phase (T2) and spotted on microscope slides coated with a thin 1% (wt/vol) agarose pad and topped with a coverslip. Samples were prepared and imaged at room temperature. Images were acquired with an AxioCam MRm camera (Zeiss) mounted on a Zeiss AxioImager M1 phase-contrast/fluorescence microscope. GFP fluorescent images were taken with a 63× air or a 100× oil objective with a numerical aperture (NA) of 1.3 using filter set 10 (Zeiss). GFP was excited at a wavelength of 450 to 490 nm, and the fluorescence was collected in the range of 515 to 565 nm.

Dual time-lapse DIC and fluorescence imaging was performed on a Zeiss Axioplan 2 as described [21 (link)]. The motorized filter wheel, two external shutters, and the 1,392 x 1,040 pixels 12-bit Photometrics CoolSNAP ES2 were controlled by μManager [22 ]. Images were acquired with an exposure time of 10–100ms for the DIC and 300 ms for the fluorescence channel using the Zeiss Filter Set 10. Embryos were allowed to develop under the coverslip without imaging and snapshots were taken at the indicating times.