Gatan orius 200

Maize pollen development analyzes were performed on epifluorecence microscope Imager M2 Axio (Zeiss®), harboring a lamp LED X-Cite® 120LED (Excelitas®). Objectives X20 and X40 were used (Plan-ApoChomat, Zeiss). Confocal imaging of maize pollen marked with both mCitrine and mCherry fluorescent reporters was done on a Leica SP8 up-right confocal microscope, with a water immersion objective (HCX IRAPO L 25x/0.95 W). Fluorophores were excited using Led laser (Leica Microsystems, Wetzlar, Germany) emitting at wavelengths of 514 nm for mCitrine and 552 nm for mCherry. Images were collected at 521-550 nm for mCitrine, 610-650 nm for mCherry. Arabidopsis root cells and pollen grain observations for both Arabidopsis and maize were done on an inverted Zeiss LSM710 confocal microscope mounted on AxioImager Z2. Samples were observed using a X40 oil objective (Plan-Apochromat 40x/1.4 Oil DIC M27, Zeiss). Dual-colour images were acquired by sequential line switching, allowing the separation of channels by both excitation and emission. Depending on the fluorophores observed, different wavelengths of excitation and band pass filters were used: mCitrine, YFP, VENUS: 514 nm / 520-580 nm and DAPI: 405 nm / 410-480 nm (excitation / band pass). Immunogold labelling imaging was done with a TEM (transmission electron microscopy) Philips CM120 at 120 kV using a CCD camera Gatan Orius 200.