Cfp 2432a

Whole plant organs were mounted under glass coverslips and observed under an epifluorescence microscope (IX70 [Olympus, Tokyo, Japan], equipped with ORCA-ER [Hamamatsu Photonics]) using 60× (numerical aperture [N.A.] 1.20, water immersion), 60× (N.A. 1.35, oil immersion), and 100× (N.A. 1.40, oil immersion) objective lenses (Olympus). Stroma-targeted GFP was detected with a filter cube U-MWIBA (Olympus; excitation: 460–490 nm; emission: 510–550 nm). FtsZ1–GFP and chlorophyll autofluorescence were detected as described previously (Fujiwara et al., 2009 (link)). CFP was detected with CFP-2432A (Semrock, Rochester, NY, USA; excitation 426–450 nm; emission 465–501 nm). To avoid rapid photobleaching of fluorescent proteins and to minimize photoresponses of plant cells, samples were observed at 6–25% excitation strength. No chloroplast photorelocations were observed during microscopy. Digital black-and-white images were imported into RGB channels of Adobe Photoshop CS3 to obtain the final merged images. To obtain line profile data, original images of CFP and GFP were aligned with ImageJ plugin StackReg, available at http://bigwww.epfl.ch/thevenaz/stackreg/. Noise reduction was performed by band-pass filtering using KBI plugins.