Deltavision core wide field microscope

Colony biofilms were grown as described before (7 (link), 36 (link)) and harvested as previously described for flow cytometry (3 (link), 22 (link)). Two microliters of the cell suspension was spotted onto a 1.5% agarose pad, and images were acquired using a DeltaVision Core wide-field microscope (Applied Precision) mounted on an Olympus IX71 inverted stand with an Olympus ×100 (NA, 1.4) lens and Cascade2 512 electron-multiplying charge-coupled device (EMCCD) camera (Photometrics). Data sets (512 by 512 pixels with 13 z sections spaced by 0.2 μm) were acquired with DIC and fluorescence optics. YFP was imaged using a 100-W mercury lamp and an FITC filter set (EX wavelength, 490/20 nm; EM wavelength, 528/38 nm) with an exposure time of 100 ms. DIC images were acquired with an LED-transmitted light source (Applied precision) at 32% intensity and exposure times between 25 and 50 ms. Postacquisition data sets were rendered and analyzed using OMERO software (http://openmicroscopy.org).

Five milliliter overnight cultures were diluted 1:200 in 25 mL minimal media and grown for 5 hr with shaking. Two microliters of culture were placed on a microscope slide layered with a pad of minimal media solidified by the addition of 1.5% UltraPure agarose (Invitrogen) and sealed with 1.5 thickness coverslips attached to the microscope slide with a GeneFrame (Thermo Fisher Scientific). Images were acquired using a DeltaVision Core wide-field microscope (Applied Precision) mounted on an Olympus IX71 inverted stand with an Olympus 100X 1.4 NA lens and a CoolSnap HQ2 camera (Photometrics), with differential interference contrast (DIC) and fluorescence optics. Images were acquired with the following parameters: 512 × 512 pixels, one-by-one binning, with 11 Z sections spaced by 0.25 μm. GFP and mCherry were detected using a GFP filter set (Ex 485/20 nm, Em 530/25 nm) and a mCherry filter set (Ex 542/82, Em 603/78), with exposure time of 100 ms. DIC images were acquired at 32% intensity and exposure time of 200 ms. Post-acquisition, images were deconvolved using softWoRx and stored and processed using OMERO software (Allan et al., 2012 (link)).