Fv1000 microscopy

Root tissues were visualized using Nomarski optics under an Olympus BX60 microscope with a dry 40× objective and photographed with an Evolution MP COLOR camera of Media Cybernetics. Confocal images were acquired using the Olympus FV 1000 microscopy with an oil immersion 40× objective.

To make Tol2-ribeye-ChR2-CFP plasmid, the promoter of zebrafish ribeye a was excised from a ribeye1.8 plasmid with NaeI and EcoRI restriction enzymes, and then cloned into the StuI and EcoRI sites of Tol2-UAS-ChR2-CFP plasmid. For the generation of Tg(Ribeye:ChR2-CFP) fish lines, the mixture of 25 pg DNA and 25 pg transposon mRNA was first injected into one-cell stage embryos, which were raised to adulthood for further screening of fluorescent signalling. F2 generation was used in optogenetic experiments. For optogenetic stimulation during calcium imaging, a 0.5 s pulse of 440-nm laser was applied to ChR2-expressing BC ATs within a circle (20 μm in diameter), while time-lapse images of calcium activities were simultaneously collected with a 900 nm or 488 nm laser via an Olympus FV1000 microscopy. In general, the volume of single BC ATs is about 3.14 × 2² × 3 μm³. On the basis of the scattering of light in larval zebrafish brain's tissues55 (link), it is speculated that around 50–100 BC ATs within a volume of 3.14 × 10² × 10 μm³ (the depth of z axis is about 10 μm) were activated during the optogenetic activation.

Cells were plated in a 35 mm imaging dish and incubated overnight at 37 °C in 5% CO₂. For the nuclear and mitochondrial staining, the cells were incubated with 200 nM Mitotracker Green and 5 μg/ml Hoechst 33,342 for 30 min in PBS at 37 °C in 5% CO₂. After washed the cells twice with PBS, 10 mM TMRE was added in a fresh culture medium. For the in vivo microscopy, the cells were maintained in the Chamlide chamber (Lice cell instrument, Seoul, Korea) and images were capture using Olympus FV1000 microscopy (Center Valley, PA, USA). The Z-stack was transformed into maximal intensity projection. The image analysis was performed using ImageJ software (24 (link)).