Plan fluor 20x

Studies of interactions between flowing bacteria and surfaces modified with poly(ethylene glycol) (PEG) brushes were conducted in a custom-built flow cell system in which the test surface comprised one wall of the flow chamber. The microscope was oriented horizontally, and the flow cell was oriented perpendicular to the floor on an optical bench to prevent gravity from contributing to cell-surface interactions. The objective used for these studies was a Nikon Plan Fluor 20x with a numerical aperture of 0.5, having a depth of field of approximately 3.5 µm. Bacteria were flowed across the surface at a shear rate of 15 s⁻¹ for approximately 10 minutes. Data was recorded at 30 fps and analyzed at a rate of 5 fps using FFmpeg software. Manual tracking was conducted using FIJI is just ImageJ. From the positions of individual bacteria in stacks of video frames, velocities of near surface cells were calculated. Further analysis and interpretation follows procedures and modeling that has been described previously,^{12 (link)} with key features explained at appropriate parts of the results.
Unless otherwise indicated, all studies employed at least 3 separate cultures for each bacterial strain, grown on separate days. These data were always sufficiently reproducible that data were combined in comparisons from one strain to another.

Microscopy studies used a Nikon TiE inverted epifluorescence microscope equipped with Nikon Perfect Focus System, Xenon arc lamp, Sutter Lambda 10-3 filter controller and Hamamatsu Orca Flash 4.0 CMOS detector. Images were collected at 97.5 fps, unbinned, at 0.65 μm/pixel (Figures 2,3) or binned 2×2 to 1.3 μm/pixel (Figure 4). All studies used a Nikon Plan Fluor 20X, NA 0.75 multi-immersion objective, water immersion mode. Since the STAFF macro measures microvascular flow in a fixed set of regions, organ stability is crucial. Organ immobilization methods for intravital microscopy are described in our previous publications^{2 (link),4 (link)}. Field of view may shift briefly during respiration, but returns to its original position after each respiration, allowing reliable measurements.