Zen 2.3 lite blue edition

Post-acquisition image processing was performed with the Zen 2.3 lite blue edition (Zeiss) and ImageJ ((http://rsb.info.gov/ij/)). Image analysis was undertaken using the ImageJ analysis program and the PSC co-localization plug-in^{56 (link)} to calculate co-localization and to produce scatter plots as described before^{11 (link)}.

The area staining for the ROS HP in B. napus cotyledons infected by Lm isolate 12CC09-GFP was measured initially at 7 dpi. Images were collected with a Zeiss Stereo-Lumar epifluorescence microscope, equipped with a NeoLumar S 0.8× objective and an Axiocam 512 camera. Light was provided by a KL-2500 LCD (bright field) or a HBO100 mercury (fluorescent images) bulb. Subsequently, the detached cotyledons were placed in a solution of DAB at room temperature. After 40 (first two experiments) or 90 (third experiment) min, the cotyledons were vacuum infiltrated with DAB for approximately 2 to 3 h. Samples were then boiled in 95% ethanol for approximately 10 to 20 min at 70 °C, to remove the chlorophyll, making the DAB staining more visible. The cotyledons were stored in 95% ethanol, prior to measurement of the area stained brown for HP under a dissecting microscope.
The lesion size, the area colonized by GFP-tagged Lm hyphae, and the area of DAB staining was measured with the aid of ZEN 2 pro or ZEN 2.3 lite (blue edition, © Carl Zeiss Microscopy GmbH, 2011) software, which automatically accounted for magnification.

Positively stained cells were manually counted and tubular, and tissue areas were measured using Zen 2.3 lite Blue Edition (Carl Zeiss Microscopy, GmbH) software. The interstitial area was obtained by subtracting the tubular area from the tissue area. All tubules per tissue section were included in the analysis. Cell numbers per tubular or tissue area (mm²) were calculated and plotted. Percentage in cell number in comparison to control was also calculated. Data were presented as mean +/− SEM. Two-way ANOVA statistical analysis was performed in order to account for inter-individual sample variation using GraphPad Prism 8 software (La Jolla, CA, USA). Statistical significance was set to p < 0.05.

The piece of amber was temporarily mounted on coverslips using glycerine. The specimen was observed under two different microscopes: An Axio Zoom.V16 with a PlanNeoFluar Z 1.0x (Carl Zeiss Microscopy GmbH) was used for the overview images and the images were saved as CZI files. For observations and for measurements ZEN 2.3 lite (blue edition) (Carl Zeiss Microscopy GmbH) was used. For higher magnifications, an Olympus IX81 inverted fluorescence microscope with UIS2 objectives, equipped with an ORCA-AG monochromatic 12-bit CCD camera (Hammatsu) was used. The mirror images were superimposed with Cell $\stackrel{ˆ}{}$ R software (Olympus Soft Imaging Solutions). Sets of photographs were analyzed with Fiji (Schindelin et al., 2012 (link)).
Single images were exported with ZEN 2.3 lite or Fiji respectively. Some images were combined with Zerene Stacker (Zerene Systems LLC, Richland, USA). The photographs were processed using Adobe Photoshop® CS6 (Adobe System Incorporated, San Jose, CA, USA) and arranged as plates. Adobe Illustrator® CS6 (Adobe Systems Incorporated, San Jose, CA, USA) was used for the lettering of the plates. Image stacks of the Olympus IX81 microscope were used for the drawings and description.