Eos utility software

For whole-mount confocal analysis, stained embryos were mounted dorsal side down in PBTriton in Attofluor cell chambers (ThermoFisher A7816), using a small fragment of broken coverglass with small dabs of vacuum grease (Dow Corning) to mount the embryo on a #1.5 coverglass (Dow Corning). Embryos were then imaged by inverted confocal microscopy on either a Zeiss LSM700 equipped with a Plan-NeoFluar 40x/1.3 oil immersion objective, or a Leica SP8 equipped with a HC PL Apo 40x/1.3 oil immersion objective. Images were captured by tile-based acquisition of contiguous z-stacks of 50–150 μm depth with 0.9–1.2 μm optical slices and 0.3–0.5 μm z-steps. Tiled images were computationally stitched together with 10% overlap per tile using Zen (Zeiss) or LAS-X (Leica) software, resulting in visible seams in some images. Maximum-intensity projections of the entire z depth were created for analysis in the same software. For confocal imaging of cryosections, slides were imaged on an inverted Zeiss LSM700 equipped with a Plan-Apochromat 20x/0.8 air objective. Z-stacks of 10–14 μm depth were imaged with 1.8–2.0 μm optical slices and 1.0–1.2 μm z-steps. For bright-field imaging, embryos were imaged in PBTriton on a Zeiss Stemi 508 stereomicroscope equipped with a Canon EOS DSLR camera and EOS Utility software (Canon).

Most of the material treated here derives from the collection of the Musée Royal de l’Afrique Centrale (MRAC), Tervuren, Belgium, with only a few duplicates retained for the collections of the University of Yaounde 1 (UY1), Cameroon or donated to the Zoological Museum, State University of Moscow (ZMUM), Russia, as indicated below. The samples are stored in 70% ethanol. Specimens for scanning electron microscopy (SEM) were air-dried, mounted on aluminium stubs, coated with gold and studied using a JEOL JSM-6480LV scanning electron microscope. The colour pictures were taken using the focus stacking setup described by Brecko et al. (2014) (link). Canon EOS Utility software was used to control the camera. Zerene Stacker was applied for stacking the individual pictures into one ‘stacked image’.
The abbreviations used to denote gonopodal structures are explained directly in the text and figure captions.

For light microscopical observations, a CKX41 inverted microscope (Olympus, Tokyo, Japan) with 1000x total magnification, was equipped with an EOS 250D camera (Canon, Tokyo, Japan). The camera was operated via EOS utility software (Canon, Tokyo, Japan).

All the images were captured using a SLR camera (Canon EOS 1000D) with 53 mm focal length and 1/15 s exposure time. Two 60 W incandescent light bulbs were used to illuminate the samples from each side. The distance between the lens and the samples was 35 cm. Due to the high resolution of the imagery device (3888 × 2592 pixels), three shoots were placed on a spectralon white platform (SphereOptics) and imaged together in order to enhance the contrast between the foreground and background. The images were captured using EOS utility software (Canon) and saved as JPG files. Individual shoots were cropped from each image and due to small variations in the size of shoots, the resolution of the images varied from 43 × 754 to 282 × 839 pixels. All the images were saved and processed on a Dell desktop computer (Intel^® Xeon(R) CPU X5560 @ 2.80 GHz × 16). The automated image analysis software was written in C++ [51 ] utilising the OpenCV Library [52 ] on an Ubuntu 14.04 operating system.

To visually observe the difference in grains formed by M. mycetomatis in G. mellonella in the presence of melanin inhibitors, four to five larvae from each group were sacrificed on the third day post infection, fixed in 10% buffered formalin, dissected longitudinally into two halves with a scalpel and processed for histology. Sections were stained with hematoxylin and eosin (HE) and Grocott methanamine silver. Grains were magnified 40x and visualized on the computer screen using the supplied EOS Utilitysoftware (Canon Inc). Grains were categorized into large, medium or small sizes using the enlargement display frame present in the Live View Shooting mode and manually counted under a light microscope mounted with a Canon EOS70D camera (Canon Inc.) by two independent scientists as previously described.^{41 (link)} The sum of all large, medium and small grains present in larvae was used to represent the total number of grains in the larvae. To estimate the total size of grain present in the larvae, the sum of all grains in a larva was multiplied by the minimum size of their respective category (large: 0.02 mm², medium: 0.01 mm² and small: 0.005 mm²).

Evaluation of GFP expression over time was achieved through the use of whole plant vacuum-agroinfiltration assays (as described by Chialva et al., 2018 (link)) along with gene transfer experiments in ‘Thompson Seedless’ somatic embryos. The GFP reporter gene expression was evaluated by epifluorescence microscopy in leaves at 1, 3, 5, 8, 13, 16, 19, and 23 days post-infiltration (dpi) (Figure 1A) and in embryogenic callus between 4 and 47 dpi (Figure 1B). Samples were observed using a Zeiss Axioscope Lab A.1 epifluorescence microscope equipped with Filter Set 09 (BP 450–490 nm) and Filter Set 38 (BP 470/540 nm; Zeiss, Oberkochen, Germany). The light source was a 470-nm LED lamp. Images were acquired with a Canon Rebel T3 camera using EOS Utility software (Canon Inc., Tokyo, Japan). The green channel was quantified via eight images per point using ImageJ software (National Institutes of Health, United States). Data was subjected to one-way ANOVA test with a 5% level of significance using Statgraphics Centurion XV (Manugistics Inc., Rockville, MD, United States).

An imaging rig was constructed (80 × 70 × 133 cm) to allow simultaneous root and shoot imaging. Cameras were fixed 1 m from the rhizotron surface and 65 cm above the plant canopy. Images were taken with an 18-megapixel full-frame digital single-lens reflex camera (Canon; EOS 1200D) equipped with an 18–55 mm lens (Canon EFS). Illumination was provided by LED-panels with constant illumination. Two cameras were controlled remotely by one laptop with EOS Utility software (Canon, USA Inc., Lake Success, NY) to trigger simultaneous image capture. The minimum detectable size of the colour 24-bit RGB image was ~ 0.1 mm pixel^− 1. The resolution of images (230 μm per pixel) could distinguish fine scale strawberry roots. Root and shoot images were taken simultaneously over 6 time points between 7 and 21 days after plant establishment.