Eclipse ti2 e inverted microscope

Imaging was performed using 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). The 40× Plan APO objective with a numeric aperture of 0.9 was used for all imaging. Fiji was then used to display the images.

For all visualizations, we used an 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). For low magnification images, we used a 20x water immersion objective with N.A. of 0.95, while for all the others we used a 60x water immersion objective with a N.A. of 1.20. We used Imaris (Bitplane) for three-dimensional rendering of z-stack pictures and Fiji for the display of all the other images.
To obtain the deformation profiles, z-stacks of the hydrogel containing fluorescent microparticles were performed every 0.5 µm, while a brightfield image of the base of the biofilm was taken to allow measurement of the diameter of the biofilm. For the visualization of the full biofilm, z-stacks of the samples were taken every 2–3 µm. For timelapse experiments, biofilms were imaged as soon as the flow was started, while for all the other experiments biofilms were imaged between 10 and 48 hr post-seeding.

Overnight bacterial cultures of PAO1 were diluted 1:1,000 in fresh LB and grown until midexponential phase. Bacterial cultures were diluted to reach an optical density of 0.05. We then loaded 6.5 μL of the diluted bacterial culture in the big channels (2 mm wide and 350 μm high) from the outlet port. It is important that injected cells do not reach the well of the bubble trap. We let the cells adhere for 30 min before starting the flow. The biofilms were grown at 25°C at a flow rate of 10 μL · min⁻¹. For time-lapse visualizations of early-stage biofilm formation, we acquired images every 15 min for 15 h in phase contrast with a 40× Plan APO NA 0.9 objective. For the visualization of biofilms, 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 sCMOS camera (Photometrics). We used a 20× water immersion objective with N.A. of 0.95, and Z stacks of the biofilms were taken every 2 μm.