Fluoview fv1000 laser confocal microscopy system

Arabidopsis siliques were observed under a Quanta 250 FEG scanning electron microscope (Thermo Fisher, Hillsboro, OR, USA). For the observation of the embryo sac, flower buds were sequentially fixed twice in methanol (5 min each), three times in ethanol (5 min each), and then in Hoyer’s solution for at least 2 h [28 (link)]. Pistils were dissected and observed as described using a FLUOVIEW FV1000 Laser Confocal Microscopy System (Olympus, Tokyo, Japan) [4 (link)]. The fluorescence signal of infiltrated tobacco leaves was detected using the same confocal system. The EYFP fluorescent was excited with 488 nm laser and the emitted light was recorded from 500 to 530 nm laser. The mCherry fluorescent was excited with 543 nm laser, recorded from 580 to 620 nm. The 543 nm laser excitation and 680 to 720 nm recording range were used for chlorophyll autofluorescence observation [18 (link)].

The 35S:PSA2-GFP-transfected protoplasts were cultivated in the dark for 12 h and then observed using a FLUOVIEW FV1000 Laser Confocal Microscopy System (Olympus, Tokyo, Japan) (Lee and Hwang, 2011 ). For transmission electron microscopy (TEM) analysis of chloroplast ultrastructure, leaves from 4-week-old seedlings were fixed, embedded and sectioned according to Faso et al. (2009) (link). A Hitachi-7700 transmission electron microscope was used for observation and image capturing.