Tie epifluorescence microscope

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).

To image AirGels at low magnification (Fig 1C and 1D), we used a Nikon TiE epifluorescence microscope equipped with a Hamamatsu ORCA Flash 4 camera and either a 10× objective with N.A. of 0.25 or a 4× objective with N.A. of 0.1. For full channel cross–sectional imaging (Fig 1E), we used a Zeiss Lightsheet Z1 dual–sided SPIM. It was equipped with PCO Edge 5.5 cameras and a 5× magnification objective with N.A. of 0.16. All the other visualizations were acquired with a Nikon Eclipse Ti2–E inverted microscope coupled with a Yokogawa CSU W2 confocal spinning disk unit and equipped with a Prime 95B sCMOS camera (Photometrics). We either used a 20× water immersion objective with N.A. of 0.95 or a 40× water immersion objective with N.A. of 1.15. We used Imaris (Bitplane) for 3D rendering of lightsheet z–stack pictures and Fiji for the display of all the other images [66 (link)].

For widefield visualizations, we used a Nikon TiE epifluorescence microscope equipped with a Hamamatsu Orca Flash 4 camera and an oil immersion 100× Plan Apo numerical aperture (NA) 1.45 objective.
For all time-lapses and mammalian cell visualizations, we used a Nikon Eclipse Ti2-E inverted microscope coupled with a Yokogawa CSU W2 confocal spinning disk unit and equipped with a Prime 95B scientific complementary metal oxide semiconductor (sCMOS) camera (Photometrics). For time-lapses, we used a 40× objective with an NA of 1.15 to acquire z-stacks with 2-μm intervals over 6 μm. Each plane was acquired at low laser power for 200 ms, allowing us to threshold out free bacteria in flow from bound bacteria. For stained mammalian cell visualizations, we used a 100× oil immersion objective with an NA of 1.45 to acquire z-stacks with 0.5-μm intervals.
We used NIS Elements (Nikon) for three-dimensional rendering of z-stack pictures.