Hc pl apo cs2 40 1.10 water immersion objective

Transparent circular microwells were used to orient embryos and image them with a confocal microscope. For this, a homemade array of little pillars made in PDMS was used to cast circular microwell in a polymer of the same refractive index as water (MY-134, MY Polymers) in a glass bottom Petri dish (P35G-1.5-14C, MatTek). For 3D reconstruction, images of 1.48 µm³ voxel size were acquired every 2 min on an inverted Leica TCS SP5, equipped with a HC PL APO CS2 ×40/1.10 water immersion objective (Leica), a Hybrid detector and a homemade cooling stage set at 19°C. The images were processed with Ilastik software to segment the membrane signal followed by an intensity threshold applied with ImageJ (Yasuo and McDougall, 2018 (link)). 3D reconstruction was performed on the segmented images with Imaris 9.0 (Bitplane), which provided measurement of the cell volume and cell sphericity. The relative animal daughter cell volume corresponds to the volume of the animal cell divided by the total volume of the daughter cells (animal and vegetal).

Longitudinal cross sections of 60 µm from transgenic nodules were prepared with a Leica vibratome VT1000S. During the procedure, nodules were glued onto a plate using super glue and kept in 50 mM NaH₂PO₄ x Na₂HPO₄ (pH 7.5). Images of the cross sections were acquired using a Leica TSC SP8 microscope equipped with an HC PL FLUOTAR 10 × 0.30 dry or HC PL APO CS2 × 40 1.10 water immersion objective (Leica, Germany) and processed using the Leica Application Suite X (Leica Microsystems). GFP and mNeonGreen were excited with a laser at 488 nm and the emitted fluorescence was collected from 502 to 537 nm. Xanthine fluorescence was collected from 595 to 622 nm after excitation at 552 nm. To prevent crosstalk between GFP and xanthine, images were acquired by sequential scanning.

Nicotiana benthamiana leaves were used for transient Agrobacterium (Rhizobium radiobacter)-mediated (co-)-expression of the constructs X131 for production of XMPP-YFP and V108 for production of mTFP1 as a cyan fluorescent cytosolic marker. Abaxial leaf surfaces were analyzed using a Leica TSC SP8 microscope equipped with an HC PL APO CS2 ×40 1.10 water immersion objective (Leica, Germany). To prevent crosstalk, images were obtained by sequential scanning with an excitation of 448 nm for mTFP1 (emission 465–495 nm) and 514 nm for YFP (emission 524–539 nm). Images were processed using the Leica Application Suite Advanced Fluorescence software (Leica Microsystems).