Las af 3

To detect PIN3-GFP, PIN4-GFP and PIN7-GFP signals in the tip of individual LRs, a binocular fluorescence microscope (Leica MZ FLIII; Leica Microsystems, Wetzlar, Germany) was used (GFP2 filters set: excitation filter 480/40 nm, barrier filter 510 nm) in combination with a DC 500 camera (Leica Microsystems, Wetzlar, Germany). To detect DR5::GFP and assess the asymmetry of PIN7-GFP signals, confocal microscopy was performed using a Leica DM6000 CS, 347 TCS AOBS (Leica Microsystems, Wetzlar, Germany) confocal laser scanning microscope, equipped with a HCX PL APO CS 348 20.0 0.70 IMM UV objective (Leica Microsystems, Wetzlar, Germany); excitation 488 nm, emission peak 509 nm). GFP signals in confocal images were analyzed and quantified using the software LAS AF 3.1 (Leica) and ImageJ (http://rsb.info.nih.gov/ij/; version 1.45j, NIH, Bethesda, MD, USA). All experiments were performed at least three times.

For live cell imaging, wherever possible, roots were mounted in a propidium iodide (PI) solution (0.02 mg/ml) for counterstaining the cell walls. MDY-64 and FM4-64 staining was performed as described (Scheuring et al., 2015 (link)). For 3D imaging, epidermal cells were recorded with a step size of 1 µm with approximately 17–20 single images. For image acquisition a Leica DM6000 CS, TCS AOBS confocal laser scanning microscope was used, equipped with a HCX PL APO CS 20.0 0.70 IMM UV or a HCX PL APO CS 63.0 × 1.20 WATER objective. Fluorescence signals of GFP (excitation 488 nm and emission 500 nm–546 nm), YFP (excitation 514 nm and emission 525 nm–578 nm), RFP (excitation 561 nm and emission 574 nm–618 nm), propidium iodide (excitation 561 nm and emission 577 nm–746 nm) and MDY-64 (excitation 458 nm and emission 465 nm–550 nm) were processed with the Leica software LAS AF 3.1 or with ImageJ (http://rsb.info.nih.gov/ij/) and data was statistically evaluated by Student's t-test using graphpad (http://www.graphpad.com/quickcalcs/). Representative images are shown.

Confocal microscopy was performed using a Leica SP5 (Leica). Fluorescence signals for GFP (excitation 488 nm, emission peak 509 nm), mScarlet-i (excitation 561 nm, emission peak 607 nm), mTurquoise (excitation 434 nm, emission peak 474 nm) and propidium iodide (PI) staining (20 µl ml⁻¹) (excitation 569 nm, emission peak 593 nm) were detected with a ×40 or ×63 (water immersion) objective. The fluorescence signal intensity (mean gray value) of the presented markers was quantified using the maximum projections obtained from a Z-stack series that were taken and analyzed using the Leica software LAS AF 3.1. The same region of interest (ROI) was defined for each individual seedling.
To determine meristematic cell numbers and cell size in stage II LRs, 8-day-old seedlings (n = 10–15) were stained with PI and microscopy was performed using confocal microscope (see above). The first two epidermal cells (adjacent to the main root) were considered for cell size measurements in stage II LR. Epidermal cell numbers were counted between the quiescent center and the first elongating cell (twice as long as wide) at the upper and lower flank of stage II LRs.
Graphpad Prism software was used to evaluate the statistical significance of the differences observed between control and genotype/treatments (one-way ANOVA).

All live recordings were captured in an ambient temperature of 21–23°C. Bright-field and epi-fluorescence time-lapse videos were recorded on a Zeiss Axiovert 200 M inverted microscope with Zeiss AxioCam MRm digital camera using AxioVision 4.8 software. High resolution images were captured with a Leica TCS SP8 confocal laser scanning microscope equipped with HCX-PL-APO-CS 10x/NA0.40, HC-PL-APO-CS 20x/NA0.75, 40x/NA1.30, 63x/NA1.20, HC-PL-APO-CS2 100x/NA1.4 oil-immersion objectives, and HCX-IRAPO-L 25x/NA0.95, HC-PL-APO-CS2 63x/NA1.20 water-immersion objectives and resonant scanning system using Leica LAS AF 3.2 software.

For the analysis of the mitochondrial position, aneuploid blastocysts were stained by MitoTracker Red CMXRos (Invitrogen, Waltham, MA, USA) and DAPI (Invitrogen, Waltham, MA, USA) with a standard protocol. The specimens were imaged with a confocal laser scanning microscope (Leica TCS SP8X, Wetzlar, Germany) with a 20× lens and oil and analyzed using LAS AF 3.2 software (Leica, Wetzlar, Germany).

To assess mitochondrial content, MII oocytes and aneuploid embryos from the same patients were stained by MitoTracker Red CMXRos (Invitrogen, USA), which incorporates into active mitochondria. DNA contents were monitored by Hoechst 33342 (Invitrogen, Ipswich, MA, USA) with a standard protocol. The specimens were imaged with a confocal laser scanning microscope (Leica TCS SP8X) with lens 20× oil and analyzed using LAS AF 3.2 software (Leica). Cell fluorescence staining allowed presentation of the localization of mitochondria and nuclei within the oocytes and embryos, and consequently to assess the distribution of mitochondria among the formed blastomeres.

Samples of ER2566 bacterial cultures (0.1 ml) bearing a gfp-tagged reporter were studied with fluorescence microscopy. Cell membranes were stained with SynaptoRed C2 (FM4-64) fluorescent dye (Sigma) at a final concentration of 5 μg ml⁻¹ for 10 min. The DNA was visualized by staining with DAPI (4,6-diamidino-2-phenylindole) at 1 μg ml⁻¹ for 10 min. Samples were then immobilized on 1-mm 1.5% agarose pads dissolved in LB medium and visualized using a Leica DMI4000B microscope fitted with a DFC365FX camera (Leica). The following Leica filter sets were used: N2.1 (for FM4-64), green fluorescent protein (for GFP), and A4 (for DAPI). Images were collected and processed using LAS AF 3.1 software (Leica).