HT‐29 cells were seeded in glass‐bottom cell culture dish plates (Greiner) with 1.5 × 106 cells per plate and incubated for 48 hr in DMEM with 10% FBS and an antibiotic mix at 37°C in 5% CO2. Cells were then treated with either TRY or SER at a concentration of 50 μg ml−1 and incubated for 2 hr at 37°C in 5% CO2. Prior to microscopy, the treated cells were washed with DPBS, fixed with 0.4% paraformaldehyde, permeabilised with 0.1% (v/v) Triton X‐100, stained with ActinGreen™ 488 ReadyProbes® (Thermo Fisher) for 30 min and washed again three times with DPBS. Confocal microscopy was performed using a Zeiss Axio Observer Z1 LSM800 equipped with an Airyscan detector and C Plan‐Apo 63x/1.4 Oil DIC objective (Zeiss). Images were acquired via the ZEN 2.3 image analysis software package (Zeiss). Because the changes in fluorescence intensity cannot be sufficiently distinguished simply by eye, ImageJ software was used to calculate the fluorescence intensity. We calculated the fluorescence intensity by subtracting the background signal of the selected area from the total area signal, as previously described (Gavet & Pines, 2010 ).
Zen 2.3 image analysis software package
Manufactured by Zeiss
ZEN 2.3 is an image analysis software package developed by Zeiss. It serves as a platform for processing and analyzing digital microscopy images. The software provides tools for tasks such as segmentation, measurement, and quantification of image data. ZEN 2.3 is designed to work with a variety of imaging modalities supported by Zeiss microscopes.
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3 protocols using zen 2.3 image analysis software package
1
Quantifying Actin Cytoskeleton Alterations
2
Visualizing S. aureus Cell Components
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Early exponential S. aureus cells were incubated with 2× MIC PPAP 23 at 37°C, 150 rpm. Samples were taken at 1.5 h intervals for 4.5 h and were subsequently stained with 10 μg/ml 4′,6-diamidino-1-phenylindole (DAPI, InvitrogenTM Molecular ProbesTM), 6.6 μg/ml FM 5-95 (InvitrogenTM Molecular ProbesTM), and 1 μg/ml BODIPYTM FL vancomycin (InvitrogenTM Molecular ProbesTM) for 5 min to visualize chromosome, membrane and cell wall, respectively (Sass et al., 2011 (link)). For microscopy, 0.5 μl of the stained samples were added to microscopy slides covered with a thin layer of 1% agarose. Images were acquired using a Zeiss Axio Observer Z1 LSM800 equipped with Airyscan detector and C Plan-Apo 63x/1.4 Oil DIC objective (Zeiss). Image analysis was performed via ZEN 2.3 image analysis software package (Zeiss).
3
Visualizing Bacterial Cell Wall and Membrane
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Cells were grown to the mid-exponential growth phase and washed with PBS. The pellets were then resuspended in PBS and incubated with BODIPYTM FL Vancomycin (0.25 μg/ml, Invitrogen) for cell wall staining and FM5-95 (7 μg/ml, Molecular Probes) for membrane staining for 10 min at 37°C. To remove unbound dye cells were washed twice in PBS and finally resuspended in PBS. For fluorescence microscopy, bacteria were mounted on microscope slides covered with a thin film of 2% agarose dissolved in PBS. Fluorescence micrographs were obtained using a Zeiss Axio Observer Z1 LSM 800 equipped with Airyscan detector and C Plan-Apo 63x/1.4 Oil DIC objective (Zeiss). Image acquisition and analysis were performed via ZEN 2.3 image analysis software package (Zeiss).
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