Zen 2.1 sp3 black edition software

Fluorescent labeling of the in vitro synthesized AtCHS-dsRNA was performed using the Silencer™ siRNA Labeling Kit with Cy™3 dye (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. A total of 35 µg of the labeled dsRNA (100 µL) was applied on the adaxial and abaxial leaf surface of four-week A. thaliana as described above. Fluorescent signals were analyzed 13–15 h after the foliar plant treatments by laser scanning microscopy. The whole leaves were mounted in distilled water in a Petri dish and were observed under a Zeiss LSM 780 laser scanning microscope operated in λ-mode equipped with a Plan-Apochromat 20×/0.8 and a Plan-Neofluar 40×/0.6 objectives. The excitation wavelength of an argon laser was set at 488 nm and the emission signal was registered at 20 evenly spaced wavelengths (8.9 nm apart) in a range from 500 to 693 nm by using a QUASAR detector. Finally, the resulted λ-stacks were linearly unmixed with the Zeiss Zen 2.1 SP3 (Black Edition) software. The laser scanning microscopy was carried out at the Far Eastern Center of Electron Microscopy (A.V. Zhirmunsky National Scientific Center of Marine Biology, FEB RAS, Vladivostok, Russia).

All images were acquired using a Zeiss LSM 780 confocal microscope (Carl Zeiss) and processed and analyzed with the Imaris (Bitplane, Zurich, Switzerland) and Image J (National Institutes of Health, Bethesda, MD, United States) software, the latter also being used for three-dimensional visualization and analysis of confocal stacks.
FaGlu fluorescence was examined under a Zeiss LSM 780 laser scanning microscope (Carl Zeiss, Oberkochen, Germany), operated in the λ-mode. The excitation wave length was 405 nm and the emission signal registered at 32 evenly spaced wavelengths (8.9 nm apart) from 408–693 nm utilizing a QUASAR detector [the peak of dopamine fluorescence is at 485–500 nm (Furness et al., 1977 (link))]. The results obtained were unmixed linearly with the Zeiss Zen 2.1 SP3 (Black Edition) software.