Epi fluorescence microscope bx 51

Light microscopy images were obtained with an Olympus Epi-Fluorescence Microscope (BX-51) essentially as described previously (Lu et al., 2017) (link). Samples for TEM were prepared using published procedures (Lu et al., 2017 (link); Armarego-Marriott et al., 2019) (link) with minor modifications. Briefly, leaf samples were fixed in 2.5% (v/v) glutaraldehyde in 50 mM sodium cacodylate (pH 7.4) containing 5 mM CaCl₂ for 1 h under vacuum. Fixation was continued at 4°C overnight, and followed by post-fixation with 1% (w/v) OsO₄ and 0.8% (w/v) K₃Fe(CN)₆ in 50 mM cacodylate buffer (pH 7.4) for 2 h at 4°C. After rinsing the leaf samples, en bloc staining of the tissue was performed by incubation in 2% (w/v) aqueous uranyl acetate for 2 h at root temperature. Following dehydration in acetone, embedding in Epon-812 (Science Services GmbH, Munich, Germany) was carried out using standard protocols. For electron microscopy, ultrathin sections (50–70 nm) were cut with diamond knives, contrasted with 2% (w/v) uranyl acetate and lead citrate, and examined in a Zeiss EM 912 Omega transmission electron microscope (Carl Zeiss, Oberkochen, Germany).