Microscopic Visualization of Plant Cell Structures

FM4-64 staining of root epidermal cells [13 (link), 61 (link), 66 (link), 67 (link)] and Lysotracker red-staining of ovules [39 (link), 59 (link)] were as described. Fluorescent images were captured using a Zeiss LSM 880 confocal laser scanning microscope (CLSM) with a 40/1.3 oil objective. GFP-RFP double-labeled materials were captured alternately using line-switching with the multi-track function (488-nm for GFP and 561 nm for RFP). Fluorescence was detected using a 505- to 550- nm filter for GFP or a 575- to 650-nm band-pass filter for RFP. YFP-RFP double-labeled materials were captured alternately using line-switching with the multi-track function (514 nm for YFP and 561 nm for RFP). Fluorescence was detected using a 520- to 550-nm band-pass filter for YFP or a 575- to 650-nm band-pass filter for RFP. Differential interference contrast (DIC) imaging of ovules were performed using a Zeiss Axiophot microscope with DIC optics. Image processing was performed with the Zeiss LSM image processing software (Zeiss).