Zen 2012 black edition

The cells were imaged using a Zeiss Elyra PS1 SIM a Plan-Apochromat 63x/1.40 Oil immersion objective and captured with an Andor EM-CCD iXon 885 camera with a 1.6X tube lens (all from Carl Zeiss, Canada). The DAPI channel was captured with 405 nm laser excitation, 23 mm diffraction grating and filter cube SR Cube 07. The z-stacks were captured in 91 nm sections with 50–90 z-stacks captured per image. The 3D-SIM and widefield images were reconstructed with ZEN 2012 black edition (Carl Zeiss, Jena, Germany) with the standard settings. Fifty nuclei per treatment were chosen randomly and imaged for each of the three replicates of the experiment. Image processing was performed in MATLAB (MathWorks, Natick, MA, USA). A central z-plane was manually selected and exported as TIF files. The granulometry of the DNA structure and the structure of DNA-poor space was measured with a morphological sieve applied to the error-function clipped images [59 (link)]. The coefficient of variation and the skewness of the intensity histogram over the detected region were also calculated. For statistical analyses, the distributions were compared using two-sided, two-sample Kolmogorov–Smirnov (KS) tests to determine any differences. p-values of <0.05 were considered statistically significant.

We used IMARIS.9.5 (Oxford Fundamental, UK) and Zen 2012 Black Edition (Zeiss) to analyze and process images. The filament tool used to show basal microvilli; the spot tool used to show 2‐NBDG; the surface tool used to show microvasculature surface.

The data were analyzed qualitatively based on the morphology and architecture of EPS and bacterial cells and quantitatively by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test (GraphPad Prism version 5.0, San Diego, California, USA) with type I error set as 0.05. 2D and 3D images were generated and processed using ZEN 2012 Black Edition ©Carl Zeiss Microscopy Gmbh platform. All experiments were performed in triplicate.

3D‐SIM images were recorded as described previously (Righolt et al., 2014). Briefly, all images were acquired with a Zeiss ELYRA PS1 (Zeiss, Toronto, Ontario, Canada) equipped with a Plan‐Apochromat 63×/1.40 Oil immersion objective using an Andor EM‐CCD iXon 885 camera and a 1.6× tube lens. The DAPI channel was obtained with 405 nm laser excitation, 23 μm diffraction grating and filter cube SR Cube 07. The lateral pixel size, Δx and Δy, was 79 nm in the recorded images and 40 nm in the reconstructed image, the step between z‐planes, Δz, was 91 nm. The 3D‐SIM images were reconstructed with ZEN 2012 black edition (Carl Zeiss, Jena, Germany) with the standard settings except for the regularization parameter, which was set to 10⁻³, and clipping, which was turned off. Clipping the image in the reconstruction stage was not done, because it sets the background to zero (black), hides actual image information and artificially increases the perceived resolution.

Immunofluorescence experiments were performed according to the same protocol as described for acetylation assay as and adapting previously described workflows for PMP70 (Niederstaetter et al. 2021 (link)), Caveolin-1 (Del Favero et al. 2020b (link)), Integrin beta 1 (Groestlinger et al. 2022 (link)) and TOM20 (Del Favero et al. 2021a (link)). The experiments were performed in µ-Slide 8 well Collagen IV (REF: 80,822, Ibidi GmbH, Gräfelfing, Germany), using the same cell number and volumes as the 48-well plates and blocking of the unspecific binding sites was obtained with 2% donkey serum for one hour. Images were obtained using Zeiss LSM710 laser scanning confocal microscope (ELYRA PS.1 system) with a 63X/1.46 Plan-Apochromat oil immersion objective (Zeiss Microscopy GmbH, Germany). The software ZEN 2012 Black Edition (Zeiss Microscopy GmbH, Germany) was used for analysis and quantification of the images. According to the datasets, images were quantified taking region of interest (ROIs, mean fluorescence relative units r.u.) or total cell area as reference (% area of the signal of interest/total cell area). For all the experiments, measurements were performed on at least 3 independent cell preparations (biological replicates) and quantifying n > 50 ROIs/cells out of minimum 9 randomly chosen optical fields.

The cells were imaged with a Zeiss Elyra PS1 SIM equipped with a Plan-Apochromat 63/1.4 Oil DIC M27 and captured with an Andor EM-CCD iXon 885 camera with a 1.6× tube lens (all from Carl Zeiss, Toronto, ON, Canada). The DAPI channel was obtained with 405 nm laser excitation, 23 µm diffraction grating and filter cube SR Cube 07. The lateral pixel size, Dx and Dy, was 79 nm in the recorded images and 40 nm in the reconstructed image. Cell nuclei were chosen randomly and imaged for each of three replicates of the experiment. At least 30 cells for each slide were analyzed (three experiments were performed). The image processing was performed in MATLAB (MathWorks, Natick, MA, USA). A field of view was selected, and the z-stack boundaries were defined manually. The 3D-SIM and widefield images were reconstructed with ZEN 2012 black edition (Carl Zeiss, Jena, Germany) with the standard settings. The granulometry of the DNA structure and the structure of DNA-poor space was measured with a morphological sieve applied to the error-function clipped images [33 (link)]. The coefficient of variation and the skewness of the intensity histogram over the detected region were also calculated.