4.4. Microscopy
For the analysis of the subcellular localization, a 5 μg aliquot of each plasmid DNA was transfected into rice protoplasts using PEG-mediated transfection and GFP signals from transfected protoplasts were observed using a Carl Zeiss LSM700 inverted confocal microscope and the image acquisition software ZEN 2009 Light Edition (Carl Zeiss, Oberkochen, Germany). A sGFP fluorescence was detected with 488 nm excitation and 505–530 nm emission wavelengths; the chlorophyll fluorescence was analyzed with 555 nm excitation and >650 nm emission. The fluorescence intensities of images were analyzed using a Histogram tool of the image acquisition software ZEN 2009 Light Edition (Carl Zeiss) following the manufacturer’s instructions. The mean intensities of images were normalized using a sGFP construct, and the ratios of the mean intensity were introduced using scale bars in Figure 1B and Figure 2C.