Incucyte zoom hd 2clr

CCB cells cultured in twenty-four-well plates were imaged hourly using an IncuCyte Zoom HD/2CLR time-lapse microscopy system (Sartorius) equipped with an IncuCyte Zoom 20× Plan Fluor objective (Sartorius). Images were collected in phase contrast and in the green (EGFP) and red (mCherry) channels. Stacks of images were exported in tagged image file format using the time plot function. Fluorescence analysis was adapted to set the basic analytical parameters with adaptive segmentation (https://ki.se/en/media/111654/download). The green calibrated unit (GCU) and red calibrated unit (RCU) threshold values were set at 2.0 for the analyses presented in Fig. 3. For the analyses related to Fig. 4, the GCU and RCU threshold values were set at 2.00 and 1.00, respectively. These parameters were set up based on the analysis of specimens infected with EGFP or mCherry recombinants to maximize the detection of each fluorochrome in its wavelength window without signal detection in the wavelength window of the other fluorochrome. This approach was used to ensure that cells detected as double positive represented cells coinfected by EGFP and mCherry recombinants and not artifact of fluorochrome spillover.

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).