Axio observer lsm 880

Cells transfected with fluorescent reporter plasmid were prepared for microscopy by transferring 200 µl of cells to a glass-bottom dish or glass-bottom 8-well chamber (Ibidi). Confocal microscopy was performed on a Zeiss Axio Observer LSM 880 with an Fast Airyscan detector and a 63 x/NA1.40 Plan-Apochromatic oil immersion objective (Carl Zeiss AG, Oberkochen, Germany). Confocal stacks were acquired by frame scanning in superresolution mode, and images were processed using the automated Airyscan algorithm (Zeiss).

Glass-bottom dishes for live cell imaging were prepared by corona-treating and poly-D-lysine coating as described in Booth et al. (2018) (link). Transfected cells were prepared for microscopy by pelleting 1–2 ml of cells and resuspend in 200 μl of 4/5 ASW with 100 mM LiCl to slow flagellar beating. Cells were plated on glass-bottom dishes and covered by 200 μl of 20% (w/v) Ficoll 400 dissolved in 4/5 ASW with 100 mM LiCl. Confocal microscopy was performed on a Zeiss Axio Observer LSM 880 with an Airyscan detector and a 63x/NA1.40 Plan-Apochromatic oil immersion objective.
Confocal stacks were acquired in super-resolution mode using ILEX line scanning and two-fold averaging and the following settings: 35 nm x 35 nm pixel size, 100 nm z-step, 0.9–1.0 μsec/pixel dwell time, 850 gain, 458 nm laser operating at 1–6% laser power, 561 nm laser operating at 1–2% laser power, 458/561 nm multiple beam splitter, and 495–550 nm band-pass/570 nm long-pass filter. Images were processed using the automated Airyscan algorithm (Zeiss).

Bacterial cultures were prepared as described above. Two hundred microliters of the diluted culture was seeded per well into 96-well microscopy plates with untreated surfaces (μ-Plate 96-well black; ibidi GmbH, Germany). Bacteria were grown in TB medium at 30°C without shaking for 6 h. Where indicated, the tested compounds were added to the medium during growth. After cultivation, planktonic cells were carefully removed and replaced with 200 μl of PBS. Fluorescent cells were visualized using a Zeiss Axio Observer LSM 880 inverted laser scanning microscope equipped with a C-Apochromat 40×/1.2 Water Corr-UV-VIS-IR objective and a 514-nm argon laser.