Orca flash 4 camera

For all visualizations of biofilms grown up to 48 h, we used a Nikon TiE epifluorescence microscope equipped with a Hamamatsu ORCA Flash 4 camera and a 40× Plan APO NA 0.9 objective. The full-channel images were stitched using the NIS-Elements software. All single cell level pictures presented in this work were taken 9 mm away downstream of the inlet. For the timelapse experiments (Supplementary Movies 1 and 2), we acquired images every 5 min for 24 h. To visualize 6 day old biofilms, we used a Leica SP8 confocal microscope equipped with a white laser, a 25× HC FLUOTAR NA 0.95, water-immersion objective, as well as a 63× HC PL APO NA 1.40 oil-immersion objective for high magnification z-stack acquisitions. We used Imaris (Bitplane) for three-dimensional rendering of z-stack pictures (Fig. 5b).

Confocal fluorescence images were acquired on a Nikon Eclipse Ti CSU-X1 equipped with 405, 488, 561, and 640 nm lasers. Emission was collected through 455/50, 525/36, 605/70, or 700/75 nm filters on an Andor iXon Ultra EMCCD camera using the NIS-Elements software. Images were visualized using ImageJ Fiji. Maintenance and selection of transgenic worms expressing fluorescent markers were performed using a Leica M165 FC fluorescent stereo microscope equipped with a Sola SE-V light source. Photobleaching and wide-field experiments were performed using a Leica TIRF microscope equipped with an infinity scanner using 488 nm laser excitation. Emission was collected through a GFP-T filter cube on a Hamamatsu ORCA-Flash4 camera.

Strains were streaked from −70 °C freezer stocks onto BSTSY agar plates and grown overnight at 37 °C with 5% CO₂. Single colonies were transferred to liquid culture and grown to exponential phase (OD₆₀₀ 0.2). Cells were spotted onto pads made of 0.8% SeaKem LE Agarose (Lonza, Cat. No. 50000) in BSTSY and topped with a glass coverslip. Cells were transferred to an Okolab stage top chamber to control temperature (37 °C) and gas (CO₂ 5% and O₂ 18%). Images were recorded with inverted Nikon Ti-2 microscopes using a Plan Apo 100 × 1.40 NA oil Ph3 DM objective using Hamamatsu Orca FLASH 4 camera. Images were processed with NIS Elements 5.02.01 software (Nikon). In all experiments, multiple x/y positions were imaged. Representative images were processed using the Fiji 2.1.0/1.53c software package.

Microcosms were sampled 4 days after the start of the selection experiment, and on the day of the common garden before inoculation of the common garden (day 78), each time by taking a sample of 500 µl. The common garden experiment was sampled 4 days after inoculation, extracting two samples of 250 µl each on day 82. Video recording and automated analysis were used to collect data on population densities (number of cells) and traits (bio-area, aspect ratio, and gross speed)^{67 (link),68 (link)}. We recorded 20 s videos (25 frames per second) of an effective sampled volume of 34.4 µl using a Leica M205C stereomicroscope at a 16-fold magnification and an Orca Flash 4 camera (Hamamatsu). Videos were analyzed using a customized version of the Bemovi package in R (available on https://github.com/efronhofer/bemovi^{67 (link)}).
Data output of the videos was visually inspected, and upon analysis, data entries reflecting P. aurelia or S. teres particles with a bio-area smaller than 1000 µm² were removed from the data as these reflected non-living particles that were accidentally tracked. After species identification, videos were again visually inspected to ensure the identification was performed correctly. Trait values of those videos that contained only one of the species were manually assigned to the correct species identity when needed.

MDA MB231 cells were grown overnight on poly-D lysine coated 20 mm, no. 1 cover slips (Neuvitro, Vancouver, WA) in a 24-well plate (Corning, Manassas, VA) at a density of 2 x10⁵ cells/coverslip. Cells were treated with a colloidal dispersion of 10 to 20 nm cerium dioxide (30% colloidal suspension in water produced by Alfa Aesar, Ward Hill, MA) particles at a concentration of 50 μg/mL in complete L-15 media for 72 h. Cells were washed three times with 1X PBST, fixed with 4% paraformaldehyde for 10 min at room temperature, and then permeabilized with 0.2% triton X-100 for 10 min at room temperature. Cells were washed and then stained with Hoechst 33342 (Abcam, Cambridge, MA), mounted and sealed with nail polish. Slides were imaged with Reflectance Structured Illumination Microscopy (R-SIM) (Nikon Corp., Japan) with Orca Flash 4 Camera (Hamamatsu, Japan). To image with R-SIM, a half-mirror was placed in the light path instead of the dichroic and the configuration was set up in the fourth channel using 488 nm laser. The dichroic was set to BS20/80 with all light paths set to “through”. Images were processed and analyzed using Nikon Nis Elements 5.0 Imaging Software. Nanoparticles were displayed in red and cell nucleus in blue for reflectance images. Cells were shown in black and white for differential interference contrast images.