A1r laser scanning confocal microscope

The procedure of Velikova, et al.^{30 (link)} was used to estimate H₂O₂ content using a standard curve of known concentrations and expressed as µg g⁻¹ FW.
MDA content was determined according to the methods described by Heath and Packer^{31 (link)}. One milliliter of extract was added to 2 mL of a reaction solution containing 20% (v/v) trichloroacetic acid and 0.5% (v/v) thiobarbituric acid. The solution was placed in a water bath at 95 °C for 30 minutes before transferring to an ice water bath. The solution was centrifuged at 10,000 rpm for 10 minutes and the absorbance of the supernatant recorded at 532 and 600 nm.
The method of Lutts^{32 (link)} was used to estimate electrolyte leakage (EL). Electrical conductivity (L1) was recorded with a conductivity meter (PCS Testr35). The samples were then autoclaved at 120 °C for 20 minutes and electrical conductivity recorded (L2) after equilibration at 25 °C.
The method of Yang^{33 (link)} was used to determine cell viability in plant roots. Cell viability assays can be performed with fluorescein diacetate (FDA). Fluorescence was measured at 488–494 nm using a microscope (Nikon A1R Confocal Laser Scanning Microscope, Japan).
For the cell non-viability assay, the method of Truernit and Haseloff^{34 (link)} was used. Fluorescence was recorded at 535–617 nm using a confocal microscope (Nikon A1R Confocal Laser Scanning Microscope, Japan).

The cells were washed twice with PBS and labelled at 37°C for 30 minutes with 400 nM MitoTracker Green (Ex 490 nm/Em 516 nm). Cells were then washed with PBS and incubated with Hoechst 33342 (1 μg/ml) for 10 minutes at room temperature. Fluorescence was detected on a Nikon A1R scanning laser confocal microscope (Nikon, Tokyo, Japan). Fluorescence was detected on a Nikon A1R scanning laser confocal microscope (Nikon Corporation, Tokyo, Japan). The images were analysed using an image analysis system (Image-Pro Plus, version 6.0) with the Mitochondrial Network Analysis (MiNA) toolset according to a previously article [20 (link)].

Following the ex vivo study, explanted tissues were fixed for histology. Briefly, selected organs were immersed in a 4% formaldehyde solution for 24 h to achieve chemical fixation. The samples were then dehydrated with ethanol, washed with xylene and paraffin-embedded. The samples were cut into 4 µm thick sections (for H&E staining), while one section of every two was cut into 10 µm thick sections for fluorescence imaging. The slides were then analyzed using a confocal microscope (Nikon A1R laser scanning confocal microscope) to acquire the fluorescence signals released by ICG and compare them to those from the adjacent sections stained with hematoxylin/eosin, acquired with a Virtual Slide Microscope (Olympus VS120). Throughout the process of fixation and paraffin-embedding, the samples were kept in the dark to avoid loss of the fluorescence signal. To assess the distribution of ICG fluorescence in the tissue sections, optical sectioning was performed by acquiring a series of focal planes 1 µm distant along the optical (z) axis of the microscope, using the z-scan mode available on Nikon A1R laser scanning confocal microscope. Orthogonal views were then reconstructed using ImageJ software (NIH).

For the observation of F. graminearum mycelial morphology and cellular localization, the various strains were grown in liquid or on solid CM media at 28 °C for 24 h, and microscopic visualizations were performed using a Nikon A1R laser scanning confocal microscope (Nikon, Tokyo, Japan). Conidia from the wild-type PH-1 and ΔFgap1^σ strains were stained with the cell-wall-damaging agent calcofluor white (CFW) at a final concentration of 10 μg/mL for visualization of cell walls and septa using an Olympus BX51 microscope (Olympus, Tokyo, Japan). Similarly, hyphae from the wild-type PH-1 and ΔFgap1^σ strains were stained with the fluorescent dye FM4-64 at a final concentration of 4 µM, incubated, and visualized for observation of endocytosis using the Nikon A1R laser scanning confocal microscope (Nikon, Tokyo, Japan).