CLSM and DIC microscopy time-lapse Image Data Sets

ABSTRACT: The morphological dynamics of pellicle formation was monitored by time-lapse confocal laser scanning microscopy. Samples were grown under a microscope in 12-well microtiter plates (Greiner CELLSTAR). To a microtiter well, 1250 µL of MSgg medium and 5 % inoculum were added. To determine the proportion of dead bacterial cells in the newly formed pellicles, we added propidium iodide (PI) in a final concentration of 3 μM into every well of the microtiter plate. The microtiter plate was placed into a Heating Insert (Pecon) that maintained a constant culture temperature. The cover of the carrier was set to 38.0 °C, and the lower part of the carrier for microtiter plates at 37.2 °C. CLSM was performed with an LSM 800 equipped Axio Observer Z1 (ZEISS) inverted microscope, using a 20 x magnification lens (NA 0.4). The YFP protein signal was excited with a diode laser at a wavelength of 488 nm (green laser), and the PI signal with a diode laser at a wavelength of 561 nm (red laser), and pinhole 97 μm. Three image stacks – (512 x 512 pixels) were measured for each well. Since the most interesting system dynamics was at the water-air interface and at the bottom of the microtiter wells, we have zoomed into these regions. First, we coarse sliced throughout the specimen (50 slices in steps of 60 μm), then we sampled the bottom of the well (50 slices in steps of 10 μm) and finally the water-air interface (100-150 slices in steps of 10 μm). The duration of the experiment was 65 h. The morphological events during pellicle formation, growth, and development were monitored by differential interference contrast light microscopy (DIC) microscopy that enabled the visualisation of pellicle in the wide-field mode. The sample preparation process was the same as for CLSM. DIC microscopy was also performed with an Axio Observer Z1 inverted microscope (Zeiss), using a 20 x magnification lens (NA 0.4). Three stacks were measured for each well. First, we sliced throughout the whole specimen (45 slices in steps of 60 μm), then we sliced the bottom of the specimen (50 slices in steps of 10 μm) and finally on the top of the preparation (150 slices in steps of 10 μm). The whole experiment lasted for 65 h.

ORGANISM(S): Bacillus subtilis

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PROVIDER: S-BIAD326 | bioimages |

REPOSITORIES: bioimages