Leica sp8 confocal microscope

For the observation of propidium iodide (PI) stained roots, 5-day-old seedlings grown on ½MS plates were mounted in ddH₂O containing 10 μM PI for 20 min before imaged with the Leica SP8 confocal microscope. Images were captured at 543 nm laser excitation and 578–700 nm emission. For the observation of TAMRA-SCOOP12 labeled roots and protoplasts, 5-day-old seedlings grown on ½MS plates or protoplasts isolated from leaves of 4-week-old soil-grown plants were treated with 100 nM TAMRA-SCOOP12 with or without 1 μM SCOOP12 or flg22 for 5 min in liquid ½MS or W5 solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES, pH 5.7), followed by washing with ½MS or W5 solution for three times before imaged with the Leica SP8 confocal microscope. Images were captured at 552 nm laser excitation and 570–620 nm emission. To observe SCOOP12-GFP in Arabidopsis pSCOOP12::SCOOP12-GFP/scoop12 transgenic plants, detached leaves were imaged under the Leica SP8 confocal microscope directly or 3 min after 5% sodium chloride (NaCl) treatment. Images were captured at 472 nm laser excitation and 493–547 nm emission. For all the observations, the pinhole was set at one Airy unit, and the imaging processing was carried out by using the Leica Application Suite X (LAS X) software.

Tg(actb2:nls-Eos); Tg(fli1:eGFP)^y1 embryos were collected and staged at developmental stages ranging from 4 to 18 ss in a glass bottom Petri dish that was precast using a 2% low melting agarose (Fisher Bioreagents, BP160-500) and a custom-made stamp (Idylle-lab) to created semi-spherical wells. Embryos were then staged and photoconverted with newly emerging somites directly at the bottom of the dish using a Leica SP8 confocal microscope. Recently developed somite pairs were selected by setting a region of interest, then photoconverted by applying maximum UV exposure (405 nm; 100% laser power) using a 20 x objective lens and high digital zoom (>X4) for 90 s. To validate the quality and specificity of the somite photoconversion, images were taken of each channel, eGFP (488 nm; 10% laser power) and mCherry (564 nm; 25% laser power). Photoconverted zebrafish embryos were then transferred to a petri dish with 1-phenyl 2-thiourea (PTU) media and incubated at 28.5  °C in the dark. At 32 hpf, embryos were embedded in a glass bottom petri dish covered by 1% agarose and imaged on a Leica SP8 confocal microscope using resonant scanning. Images of the dorsal aorta were taken in a sequential stack manner for eGFP (488 nm; 10% laser power) and mCherry (564 nm; 35% laser power) using a 20 x objective lens.

The animal was perfused with 4% paraformaldehyde (PFA) in chilled saline through the heart and the brain was removed from the skull. The brain was post-fixed overnight in 4% PFA then transferred to 10% sucrose until it sank. Coronal sections (100 μm) were prepared using a microtome. Sections were mounted using VectaShield, and imaged using a Leica SP8 confocal microscope. Native fluorescence image Z-stacks were collected with either a 10 × , 0.4 NA dry, 20 × 0.85 NA oil-, 40 × , 1.3 NA oil- or 63 × , 1.4 NA oil-immersion objective at 2 μm step size. For revealing morphological detail, Z-stacks were tiled to cover the dendritic arbour across adjacent slices. These stacks were stitched using an ImageJ stitching plug-in and aligned with the Vaa3d software (www.vaa3d.org). Images of the larger field of view were taken with a 10 × , 0.4 NA dry objective. For biocytin filled cells, the slices were processed with Biocytin-Streptavidin reaction then imaged under the Leica SP8 confocal microscope.