Csu22 spinning disk confocal

For imaging, Transwell filters were excised with a scalpel and mounted on glass slides using Prolong Gold with or without DAPI (Invitrogen). Unless otherwise indicated, images were acquired using a Nikon FN-1 Microscope equipped with a Nikon C1si spectral confocal, a Nikon Plan 100×/1.4 oil objective, and C1si software (Nikon). For samples stained with DDAO, which required a far-red laser, images were acquired on a Nikon Ti-E microscope equipped with a Yokagawa CSU22 spinning disk confocal and a Nikon Plan Apo VC 100×/1.4 oil objective using Micro-Manager software [52] (link). Confocal images of bacterial aggregation within murine lungs were acquired with a Zeiss UV LSM510 confocal microscope using a 100× oil objective.

We performed majority of the fixed and live imaging on Leica SP8-X inverted confocal system equipped with HyD hybrid detectors, adaptive focus control and Okolab environmental control incubator cage. Live imaging was performed on Nikon Ti inverted fluorescence microscope with CSU-22 spinning disk confocal, EMCCD camera, and incubator, CO₂, and humidity control at the UCSF Nikon Imaging Center. Images are represented using pseudocolors suitable for color-blind palette.