Ax10 imager a2

Cavity slide culture method was used for studying F. solani spore germination⁴¹ . A PDA spore suspension of F. solani at a final concentration of 1 × 10⁵ spores mL⁻¹ in sterile water were prepared 25 µL of which was mixed with an equal volume of different liquid media (Table S1) in cavity slides separately. The cavity slides were incubated in moist chamber at 28 °C and the spore germination was observed under a light microscope after 24 h. Spores mixed with sterile distilled water served as control. Upon incubation, a drop of lactophenol-cotton blue (40 mL glycerol, 20 mL lactic acid, 20 g phenol and 5 mL of 1% aqueous cotton blue) was added to each slide. The germinated and non-germinated spores were counted under a light microscope and percentage of germination was determined. The morphology of spores and their germination characteristics were observed using phase contrast microscope (Zeiss AX10 Imager A2, Zeiss, Germany) under bright field at × 40 magnification.

Detection of lipofuscin was performed as previously described [25 (link)]. The samples were examined in a laser scanning confocal microscope (Zeiss AX10 Imager A2, Zeiss, Göttingen, Germany) with sequential scanning and detection of dot-like lipofuscin granules, respectively, followed by merging and saving of the images. For quantitative analysis, from each TEM image, using the open source ImageJ software (http//:imagej.nih.gov (accessed on 20 January 2021); NIH, USA) the amount of lipofuscin in 100 μm² and fractional lipofuscin granules were measured by obtaining the area occupied by lipofuscin over the area in 100 μm² occupied by cytoplasm. One eye from each animal was included, and each eye was averaged (n = 6 animals).

The RPE layer of the stained sections was examined with a confocal microscope (Zeiss AX10 Imager A2, Zeiss, Germany) using a 63x (NA:1.42. Plan Apochromat) oil (Zeiss Immersol™, Germany) immersion objective. The microscopy settings were kept identical for all pictures taken and held constant during imaging. Representative high-power microphotos were taken close to the vicinity of the optic nerve with ZEN blue v2.3 (Carl Zeiss Microscopy, Germany). At least nine repetitive images were taken per animal, and several regions of interest were analysed per view and averaged. All the captured images were processed using ImageJ (version 1.52a). The background was subtracted using a default rolling ball radius method. Regions of interest (ROIs) were drawn followed by mean grey-value measurement. ROIs were kept constant within each antibody analysed. All the imaging analyses were blindly quantified at least by 3 independent researchers. The number of biological replicates per group varied between four and five (Table 2). Images were color enhanced using Adobe Photoshop® for visual representation.

Eye sections were examined with a confocal microscope (Zeiss AX10 Imager A2, Zeiss, Göttingen, Germany). Images were recorded sequentially from the green, red and far-red channels on optical slices of 0.1 μm of focal thickness using a 63× oil immersion objective (NA:1.42, Plan Apochromat). The microscopy settings were identical for all scans and kept constant during imaging. Representative high-power micrographs were taken with ZEN black software and processed with Adobe Photoshop. In all imaging procedures, gamma adjustment was performed on the whole image in order to maintain appropriate contrast. Representative images from mice (n = 6) were used for the demonstration of each observation.

Macroscopic and microscopic characters were observed and measured as previously described. The isolates were inoculated and incubated for 7 d on the agar media Czapek yeast autolysate agar (CYA), yeast extract sucrose agar (YES), creatine sucrose agar (CREA), dichloran 18% glycerol agar (DG18), oatmeal agar (OA), and malt extract agar (MEA; Oxoid CM0059) [4 (link)]. Colony colors were referenced to numbered color codes in parentheses [66 ]. Light microscope preparations were made from one wk old colonies grown on MEA. To induce ascomata formation, colonies were incubated for more than two weeks. A Zeiss Stereo Discovery V20 dissecting microscope and a Zeiss AX10 Imager A2 light microscope, both equipped with an Axiocam 506 color camera and ZEN v.2.0 software (made in Germany), were used to capture digital images.

The slides were mounted using the Mowiol mounting media and stored in the dark at room temperature. The stained sections were examined with a confocal microscope (Carl Zeiss AX10 Imager A2, Carl Zeiss Microscopy, Jena, Germany) using a 63× (NA:1.42. Plan Apochromat) oil (Zeiss Immersol™) immersion objective. At least nine repetitive images were taken from each section for all markers. Images were colour enhanced using Adobe photoshop for visual representation. All the captured images were converted into 8‐bit and processed using ImageJ v1.53t (https://imagej.nih.gov/ij/). The background was subtracted using a default rolling ball radius method. Regions of interest (ROI) were drawn over RPE cell layer followed by colocalization analysis using 2 channel spots colocalization analyser ComDet v0.5.5 (https://imagej.net/plugins/spots‐colocalization‐comdet), an ImageJ plugin. The total number of puncta from each colour (channel) was calculated and the corresponding correlations of colocalization were measured. The mean grey value was used to quantify optical changes seen in images.