Air objective

The biofilms were stained using LIVE/DEAD BacLight Bacterial Viability Kit for microscopy (LifeTechnologies GmbH, Darmstadt, Germany) according to the manufacturer’s protocol.
Stained biofilms were analyzed under vital conditions using an inverse confocal laser scanning microscope LSM510 (Carl Zeiss AG) by excitation at 490 nm using the argon laser line and 40 x air objective (Carl Zeiss AG). An area of approximately 100 μm (X) x 100 μm (Y) was screened in 1 μm Z-intervals (Z-stack) at green (522 nm) and red (635 nm) channels, respectively. The pinhole was adjusted to 1 μm. The biofilm data were visualized by ZEN 9.0 software (Carl Zeiss AG). The 2D images of the green and red channel (24-bit) of each experiment were exported as two-dimensional bitmap images numbered (postfix) according to their Z-layer number with a size of 1024x1024 pixels and a resolution of 72 dpi.

All imaging was done on a Zeiss LSM780 inverted confocal laser scanning microscope equipped with a diode laser for 405nm excitation of tracheal extracellular matrix autofluorescence and DAPI, an Argon laser for 488nm excitation of GFP and YFP, and a DPSS 610-1 laser for 561nm excitation of DsRed, as well as a transmission photomultiplier tube detector to detect transmitted light. For terminal cell imaging, the objective used was a Plan-Apochromat 63X/1.4 Oil DIC M27 (Zeiss). For scoring dorsal tracheal anatomy phenotypes, a 20X Air Objective (Zeiss) was used as this allows a greater imaging depth, necessary to trace the sometimes very deep dorsal anastomoses.

Imaging was performed using a high content imager (Operetta CLS). The device operates a high-speed motorized table. Images were acquired using a 20x air objective (NA = 0.4; Zeiss, Jena, Germany) and a 16-bit sCMOS camera with laser-based autofocus. Cells were stained with DAPI in images in the brightfield, digital-phase contrast (DPC), and the λ_ex = 535–585 nm and λ_em = 430–500 nm fluorescence channels were acquired. Image acquisition settings were kept constant. For each independent experiment, four technical replicates with a total of 36 fields of views per condition and experiment were imaged. For the quantification of cell counts, cell area, and morphology, an algorithm-based analysis was performed using Harmony 4.9 image acquisition and quantification software (PerkinElmer, Hamburg, Germany) after segmenting individual cells via their pseudo-cytosolic DPC signal.

Cells were fixed with 4% paraformaldehyde in PBS for 20 minutes and were then permeabilized and blocked for 1 hour with 0.3% Triton X-100, 2% bovine serum albumin, 5% donkey serum and 5% goat serum. The Spike S primary antibody (1:100, Sino Biologicals Cat# 40,150-R007) was added to samples overnight at 4°C. Samples were rinsed 5 times for 2 minutes each with PBS containing 0.3% Triton X-100. The goat anti-rabbit Alexa Fluor® 488 IgG (Abcam, Cat# ab150077) secondary antibody was diluted 1:1000 in blocking buffer and was then added for 2 hours at room temperature. Samples were then rinsed 5 times for 2 minutes each with PBS containing 0.3% Triton X-100, followed by DAPI diluted in PBS at 1:5000 for 10 minutes. Images were obtained using Zeiss LSM880 confocal microscope using the Airyscan detector and a 20X air objective (Carl Zeiss GmbH, Jena, Germany) and were quantified using CellProfiler 2.0. Alternatively, plates were imaged using the Operetta (PerkinElmer, Waltham, MA) system.

Biofilms were stained with the LIVE/DEAD BacLight Bacterial Viability Kit for microscopy (Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer’s protocol (Thieme et al., 2018 (link)). SYTO9 stains the nucleic acid of living and dead cells and propidium iodide stains only the dead cells (Peeters et al., 2008 (link)). Stained biofilms were analyzed under vital conditions using an inverse confocal laser scanning microscope (CLSM) LSM780 with a 40 × air objective (Carl Zeiss AG, Jena, Germany) at 490 nm excitation by an argon laser. An area of ca. 100 μm (x-axis) × 100 μm (y-axis) was screened in 2 μm intervals in the z-axis (z-stack) in the green (emission 522 nm) and red (emission 635 nm) channels, respectively. The biofilm images were visualized by ZEN 9.0 software (Carl Zeiss AG, Jena, Germany). The biofilm experiments [eradication (BEC) and prevention (BPC)] were independently performed in triplicate for each assay. Quantitative analysis of biofilm images was performed by an algorithm termed qBA (quantitative biofilm analysis) that determined the number of bacterial counts/cm² (Klinger-Strobel et al., 2016 (link); Thieme et al., 2018 (link)).