Incucyte zoom hd 2clr time lapse microscopy

After termination of the magnetic stirring process to exert tornadic shear stress (TSC), spore suspensions were promptly diluted in RPMI plus 2% glucose at a concentration of 1 × 10³ spores per ml. A total of 200 spores were seeded per well of a 96-well flat-bottom plate. For selected experiments, serial dilutions of hydrogen peroxide (H₂O₂; Fisher Scientific, final concentration, 0.25 to 64 mM), amphotericin B (0.03 to 16 μg/ml), or posaconazole (0.03 to 16 μg/ml) were added. Phase images were obtained hourly for 24 h at 37°C in the IncuCyte Zoom HD/2CLR time-lapse microscopy system (Sartorius) equipped with an IncuCyte Zoom 10× Plan Fluor objective (Sartorius). The IncuCyte image analysis software was used to quantify mycelial confluence, hyphal length, and branch point numbers as described before (28 (link)).

Well plates were incubated and imaged for 18 h at 37 °C in the IncuCyte ZOOM HD/2CLR time lapse microscopy system (Sartorius) equipped with an IncuCyte ZOOM 10 x PLAN FLUOR objective (Sartorius) as previously described [11 (link)]. Phase images were acquired hourly for all experiments. For live cell co-culture using AF293-GFP, green fluorescence images were obtained hourly with an acquisition time of 400 milliseconds. NT image processing algorithms were applied using our published parameters for A. fumigatus and AF293-GFP [11 (link)].