Lsm 800 airyscan microscope

Staining of cells was performed by the methods described previously15 (link). Slides were examined under a Zeiss 510 Meta Confocal Laser Scanning Microscope and LSM 800 Zeiss Airyscan microscope performed at the Advanced Microscopy Core Facility of the University of Nebraska Medical Center. Images were analyzed using ZEN 2009 software. For some figures, image analysis was performed using Adobe Photoshop and ImageJ. F-actin staining was performed using CytoPainter F-actin Staining Kit-Blue Fluorescence according to the manufacture’s (Abcam) protocol. Statistical analysis of colocalization was performed by ImageJ, calculating the Pearson correlation coefficient58 (link).

Biofilm assays were performed following the O’Toole (2011) (link) protocol. Briefly, overnight cultures were inoculated into LB at a final OD₆₀₀ of 0.1. One hundred microliters of culture was added to 96-well polystyrene plates and incubated over a 5-h period without shaking. At the indicated times, cells in suspension were removed, and the wells washed twice with distilled water. One hundred twenty-five microliters of 0.1% crystal violet was then added to the wells, incubated for 10 min, and washed three times with 125 μl of distilled water. To solubilize the crystal violet, 125 μl of 30% acetic acid was added and the absorbance measured at 550 nm. The ratio OD₅₅₀/OD₆₀₀ was calculated to normalize the biofilm formation due to variation in bacterial growth.
For biofilm imaging, bacteria were grown in BM2 minimal medium (Repizo et al., 2015 (link)) supplemented with 10 mM of potassium glutamate (BM2G) as carbon source in a glass-bottom 24-well μ-plate. Cultures were inoculated at an initial OD₆₀₀ of 0.1 from an overnight culture grown in LB and then incubated at 37°C under static conditions. At indicated times, the biofilm was labeled with DAPI, and images were taken with a confocal Zeiss LSM800 Airyscan microscope and analyzed with ImageJ and Imaris software.

MoDCs were mixed with pre-warmed RPMI-1640 medium without phenol red and incubated for 1 h at 37°C 5% CO₂. Particles were added in a 1:10 cell/particle ratio for 30, 60, 90, or 120 min and imaged with a Zeiss LSM 800 AiryScan microscope equipped with a 63× 1.4 NA (numerical aperture) oil immersion objective at 37°C.

Wide-field image montages were automatically generated in Neurolucida computer software (MBF Bioscience, Williston, VT, USA) by taking consecutive pictures with an automated stage controller mounted on a Zeiss Axio Imager M1 (Carl Zeiss, Oberkochen, Germany). Confocal micrographs were captured using a Zeiss LSM 800 Airyscan microscope with ZEN (blue edition) computer software. Light-sheet micrographs were acquired with an UltraMicroscope II (LaVision Biotec, Bielefeld, Germany) setup using Imspector computer software. All intestinal tissues were randomized and sampled consecutively with the same acquisition settings. Postacquisition brightness/contrast adjustments were performed uniformly on all light-sheet micrographs.