Eclipse te 2000 e2 confocal laser scanning microscope

Reactive oxygen species cellular localization was determined by confocal microscopy in an Eclipse TE–2000–E2 Confocal Laser Scanning Microscope (Nikon) with excitation at 488 nm and emission at 515/530 nm, after leaf staining with the ROS-sensitive fluorescent probe DCFDA. Leaf disks (1 cm in diameter) from 6 plants of each line were collected during the light period, vacuum-infiltrated in the dark with 50 μM DCFDA in 10 mM Tris-HCl pH 7.5, incubated in the dark for 1 h in the same solution, washed briefly and mounted in water. Images were acquired with a 20x objective (Plan-Apochromat 20X/0.75), image size 512 × 512 pixels, 16 bit depth. Before recording images, the signal intensity across the entire view was visually inspected in order to prevent signal saturation. Imaging was performed by scanning 7 optical slices (with an interval of ∼1 μm) of the palisade parenchyma immediately next to the epidermis. Fluorescence intensities were estimated using Fiji software (Schindelin et al., 2012 (link)). Stacks were compiled to single images (z-projections) and presented as a “sum slices” projection type. Fluorescence intensities were calculated using the z-projections.