Visiscope confocal cell explorer

Spinning disc confocal imaging of mitochondria (MT_rk or MT_gk), GFP-Miro2 fusions and ER (ER_rk) in live tobacco epidermal pavement cells was performed using a VisiScope Confocal Cell Explorer under the control of VisiView software (Visitron Systems, GmbH Germany), composed of an IX81 motorised inverted microscope (Olympus, Germany), a CSU-X1 Spinning Disc unit (Yokogawa, Japan), a PlanApo UPlanSApo × 100 (1.4 NA) oil objective (Olympus, Germany) with a Photometrics CoolSNAP HQ2 camera (Roper Scientific, Germany). To achieve dual fluorescent imaging, GFP was excited with a Sapphire 488 nm 70 mW laser and mCherry with a Cobolt Jive 561 nm 70 mW laser. All movies were taken using a temporal resolution of five frames s⁻¹, 100 frames long with a spatial resolution of 0.129 µm pixel⁻¹.

The mounted slides were rinsed twice in PBS, preincubated in 10% NGS for 30 min, and incubated with the antibody against Necab2 (1:1,000), diluted in the previous solution for 24 h. Then, slices were washed in PBS, preincubated with a 0.2% BSA/PBS solution for 30 min, and incubated with a red-fluorescently labeled secondary antibody (Alexa Fluor^® 633, Thermo Fisher Scientific Inc., Waltham, United States; RRID: AB2535732; 1:1,000) in 0.2% BSA/PBS solution. After final washing in PBS, the slides were coverslipped in anti-fade medium (Prolog Gold, Molecular Probes^®, Thermo Fisher Scientific Inc., Waltham, United States). The immunofluorescence staining was imaged with an inverted Confocal Spinning-Disc-Laser Microscope (VisiScopeConfocal—Cell Explorer, Visitron Systems GmbH, Puchheim, Germany) and the computer-assisted software cellSens Entry (Olympus Life Science Solutions, Hamburg, Germany) and photoprocessed as described above.