Photometrics prime bsi

KPL-4 cells were grown on LabTek-II eight-well slides to approximately 70% confluency and incubated with 2 µM MC1_Cy5.5 and the micropinocytic probe Oregon Green Dextran (Molecular Probes, D7171; 200 µg ml⁻¹) and/or the clathrin-dependent recycling marker Alexa 555–transferrin (12.5 µg ml^–1; Molecular Probes, T35352) for different lengths of time. Cells were then washed four times in cold medium, fixed for 20 min in 3% paraformaldehyde, washed twice for 5 min each in PBS and mounted in Prolong Gold plus DAPI (Invitrogen, P36931). Mitochondria were stained with 200 nM Mitotracker Red FM (Molecular Probes, M22425) for at least 20 min before live imaging.
Spinning disk confocal microscopy was performed using a CSU-W1 (Yokogawa) spinning disk on a Zeiss AxioObserver M1 microscope with a ×63/1.4-NA objective equipped with 405-, 488-, 561- and 640-nm lasers. Images were acquired using SlideBook v6 (Intelligent Imaging Innovations) and a Photometrics Prime BSI (Teledyne Photometrics). Figures were assembled in Adobe Photoshop 2021, and any gamma adjustments to the contrast were applied across the whole image.

The surface interaction of representative isolates that either remained attached to the surface or aerosolized well during nebulization were compared in a model of rewetting bacteria dried on a surface. Overnight bacterial cultures grown in LB at 37°C were normalized to an optical density at 600 nm (OD₆₀₀) of 2.5 and subsequently diluted 1:100 in LB. Five microliters of the suspension was carefully spotted onto the middle of a glass well in a 96-well plate (Mat Tek/P96G-1.5-5-F) and dried for 3 hours at either 16% or 70% RH. Once dried, the bacteria were loaded with the membrane dye FM4-64 (ThermoFisher; T3166) and the cytoplasm dye CytoX (ThermoFisher; S7020) 15 minutes prior to viewing. Imaging was performed with a spinning disk confocal microscope (Nikon Ti2-E connected to Yokogawa W1), and the images were captured with a sCMOS camera (Photometrics Prime BSI; Teledyne Photometrics, Tuscon, AZ) (see Supplemental Methods for more detail). The chamber was then wetted repeatedly with water followed by removal 30 times, and the same field was captured after the vigorous wash. The procedure was subsequently repeated for each sample well. Image analysis was performed with the built-in functions in the Nikon Element software.