Incucyte zoom 10 plan fluor objective

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 imaged hourly or every 2 h in the IncuCyte Zoom HD/2CLR time-lapse microscopy system (Sartorius) (18 ) equipped with an IncuCyte Zoom 10× Plan Fluor objective (Sartorius). Imaging was performed for 16 to 60 h at 37°C. Imaging periods and intervals for each individual experiment are specified in the figure legends. Phase images were acquired for every experiment. For fluorescence imaging, the acquisition times were 400 ms for the green channel and 800 ms for the red channel. Representative images capturing different morphotypes and fungal cell densities were used for training image collections (Fig. 1A). Analysis parameters for Basic Analyzer (BA; endpoint, confluence [%]) and NT (IncuCyte Zoom NeuroTrack software module; endpoints, neurite length [mm/mm²] and branch points [1/mm²]) processing definitions were optimized individually for each species and (if applicable) fluorescent labeling strategy according to the workflow outlined in the manufacturer’s manual (31 ). Ranges of key processing parameters are summarized in Table 1 (phase) and Table 2 (fluorescence). The optimized processing definitions were subsequently used for real-time image analysis (Fig. 1B).