Cell observer sd microscope

Seeds were sterilized in 30% bleach with 0.1% SDS, rinsed with sterile water, and resuspended in 0.15% agar. After stratification at 4 °C for 3 days, seeds were grown on ½ MS (2.2 g/L Murashige and Skoog salts, 0.6 g/L MES, pH 5.6), 1% (w/v) sucrose 0.8%, agar plates for 6 days. Healthy seedlings were picked to float on stomatal function assay solutions (dark-induced closure: 20 mM KCl, 1 mM CaCl₂, and 5 mM MES- KOH, pH 6.15; light-induced opening: 50 mM KCl, 0.1 mM CaCl₂, and 10 mM MES-KOH, pH 6.15). To induce opening/closure of stomata, samples were pretreated under dark/light, respectively, for 2.5 h. Seedlings were then transferred to light/dark environments, respectively, and imaged. Guard cell images were recorded using a Nikon D5100 DSLR camera and Zeiss Cell Observer SD microscope before and after induction; guard cell dimensions were measured using ImageJ.

At given time points (1, 7, or 14 days), cell viability was assessed using fluorescence-based LIVE/DEAD^TM Viability/Cytotoxicity Kit for mammalian cells (Thermo Fisher Scientific, Waltham, MA, USA); Calcein dye is maintained in living cells giving green fluorescence. In contrast, ethidium homodimer-1 in cells with damaged membranes shows red fluorescence upon nucleic acid binding. Calcein AM and Ethidium homodimer-1 were suspended in sterile PBS (2 µL of Ethidium homodimer-1, 0.5 µL of Calcein-AM in 1 mL of PBS), and 200 µL of the solution was added per well, followed by 20 min of incubation at room temperature (RT). The images were collected using a Cell Observer SD microscope (Axio Observer Z.1, Carl Zeiss, Jena, Germany) in Z-stack mode with an interval of 2.13 µm. Filter excitation/emission wavelength 488/509 (green channel) and 545/572 (red channel) were used. Ranges of emission: 450–490, 500–550, 538–562, 570–640. Image size in pixels: 1388 × 1040 with the scaling (per pixel) 1024 × 1024. Obtained images were analyzed using Zeiss ZEN (Carl Zeiss, Jena, Germany) and ImageJ software (v.1.53o).

Images were acquired using a Nikon SMZ25, a Zeiss Stereo Discovery V8, a Zeiss LSM780 confocal, or a Zeiss LSM800 confocal after embryo anesthesia with a low dose of tricaine and immobilization in 2% low-melting agarose in glass bottom petridishes (MatTek corporation). For time-lapse imaging, images were acquired using a Zeiss Cell Observer SD microscope. Embryos were mounted in 0.6% low-melting agarose and kept at 28 °C. Time-lapse images were recorded every 30 min. Facial lymphatics were measured using the ZEN software to determine the extent of formation of the different facial lymphatic vessels^{55 (link),56 (link)}.