M205 fa stereomicroscope

Video recording was conducted using a Leica Stereomicroscope M205 FA equipped with a DFC9000 GTC CCD camera (Leica Microsystems, Wetzlar, Germany) with 160-fold magnification at 250 frames per sec. Zebrafish embryos 3 dpf were anesthetized in 160 mg/L MS222 (Sigma-Aldrich) in E3 medium and placed horizontally in a small Petri dish filled with E3 medium to obtain lateral view. The left eye was facing downward for optimal imaging of the ventricle.
The GFP-positive transgenic embryos were visually phenotyped based on the recorded videos and the number of cardiac-specific defects was determined. The defects were classified as follows: looping defects, pericardial edemas, arrhythmias, altered ventricular shape, strong ventricular contractions, heterogeneous ventricular contractions, and altered atrial shape.
Images of end-diastole and end-systole of three contractile cycles were analyzed with ImageJ/Fiji (https://fiji.sc/ (accessed on 14 March 2017)). To determine end-diastolic ventricular area and end-systolic ventricular area the perimeter of the ventricle was outlined. Length of the short axis (width) was measured between the myocardial borders of the ventricle to calculate FS as follows: %FS = [(width at diastole − width at systole)/(width at diastole)]. For %FS only embryos with proper looped hearts were analyzed.

Roots or root tips from 10‐ to 11‐day‐old seedlings were placed on growth plates or, in ‘wave’ experiments, a root was placed across a gap in growth medium agar (Matthus et al. 2019c ; Fig. S1). Recovery was for 5 to 10 min. Solution (3 µl) was applied by pipette; control solution (full Pi or zero Pi liquid half MS) ± 1 mM eATP. Imaging was with a Stereomicroscope M205 FA (Leica Microsystems, Wetzlar, Germany), with a DFC365FX camera (Leica) and a Sola SE365 light source (Lumencor, Beaverton OR, USA); excitation 470/40 nm, emission every 5 s at 525/50 nm, gain of 2.0 and 30× magnification. ImageJ Fiji was used to process GCaMP3 GFP signal intensities, fitting regions of interest (Roi) with the ‘ROI Manager’ tool. Z‐axis profiles were plotted for each Roi, and background signal was subtracted. Data normalization was as described by Vincent et al. (2017 (link)): ΔF/F₀ = (F–F₀)/F₀, where F is the fluorescence signal and F₀ is the baseline fluorescence signal. Maximal response was ΔF_max/F₀. Intensiometric false‐colour videos of response to control solution or ATP were compiled from a representative time series.

Species identification was based on morphological features of the fruiting bodies, which was produced on the infected plant tissues, including stromata (arrangement and size), conceptacle (presence or absence), conidiomata (color, size, and shape), ostioles (number and diameter), locules (number and arrangement type), conidiophores, and conidia (size and shape), supplemented by cultural characteristics such as colony color, texture, and the presence or absence of airborne hyphae (Mostert et al., 2001 (link); Zhang et al., 2007 ; Zhu et al., 2020 (link)). The morphological features were observed under a Leica stereomicroscope (M205 FA) (Leica microsystem, Wetzlar, Genmany). Micro-morphological observations determined by a Nikon Eclipse 80i compound microscope. Measuring 30 conidiomata/ascomata and 50 conidia/ascospores, determined by length, width, and length/width ratio (L/W ratio). Recording the colony diameters and describing the color was based on the color charts of Rayner (1970) after 1–2 weeks on PDA in darkness. The results were edited manually by Adobe Bridge CS v. 6 and Adobe Photoshop CS v. 5.

O₂⁻ production was detected in situ by vacuum-infiltrating leaf and root samples with 0.05(M) sodium phosphate buffer containing 0.05% NBT for 15 min followed by incubation for another 45 min. Leaf tissues were subsequently transferred into absolute alcohol for de-chlorophyllization and imaging was done under a Leica stereo microscope M205FA equipped with a Leica DFC310FX digital camera (Leica Microsystems). Plant sections were studied under a bright field microscope (n = 10).

Optical determinations of heart rates were performed using a Leica stereomicroscope M205FA (Leica Microsystems, Switzerland) equipped with a Leica DFC365X camera (image acquisition software: Leica AF6000). Heart rates (HR) were extracted from 10 second-movies (15 frames per second) by optical evaluation followed by extrapolation to beats per minute.

The identification of species was based on morphological characteristics of the ascomata or conidiomata formed on infected host materials. Macro-morphological features (structure and size of conidiomata and ascomata, ectostromatic disc and ostioles) were photographed using a Leica stereomicroscope (M205 FA) (Leica Microsystems, Wetzlar, Germany). Micromorphological features (conidiophores, conidiogenous cells, asci and conidia/ascospores) were photographed using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan), equipped with a Nikon digital sight DS-Ri2 high resolution colour camera with differential interference contrast. Over 30 conidiomata were sectioned and 50 conidia were selected randomly to measure their lengths and widths. Colony diameters were measured and the colony colours described after 3 days and 14 days according to the colour charts of Rayner (1970) .

Species identification was based on morphological features of the ascomata or conidiomata from infected host materials and micromorphology, supplemented by cultural characteristics. Microscopic photographs (structure and size of stromata; structure and size of ectostromatic disc and ostioles) were captured using a Leica stereomicroscope (M205 FA) (Leica Microsystems, Wetzlar, Germany). Microscopic observations (shape and size of conidiophores, asci and conidia/ascospores) were determined under a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan), equipped with a Nikon digital sight DS-Ri2 high definition colour camera, using differential interference contrast (DIC) illumination. The Nikon software NIS-Elements D Package v. 3.00, Adobe Bridge CS v. 6 and Adobe Photoshop CS v. 5 were used for the manual editing. More than 10 conidiomata/ascomata, 10 asci and 30 conidia/ascospores were measured by Nikon software NIS-Elements D Package v. 3.00 to calculate the mean size/length and respective standard deviations (SD). Colony diameters were measured and the colony features were described using the colour charts of Rayner (1970) .