Lsm 880 axio examiner z1

The in vivo imaging of foxj1a:RFP; foxj1b:GFP larvae were performed at 5 dpf. The larvae were immobilized using tricaine methanesulfonate (MS222) and were subsequently embedded in 1.5% LMP agarose (Thermo Fisher Scientific), with the nose pointing up. After the agarose was solidified, confocal imaging was performed with Zeiss LSM 880 Axio Examiner Z1 with a Zeiss 20X plan water objective, NA 1. In vivo imaging of foxj1b:GFP; OMP:gal4; UAS:NTR-mCherry larvae was done similarly at 4 dpf.

Microscopy was performed on a Zeiss LSM 880/Axio Examiner Z1 motorised upright laser scanning microscope equipped with Argon multiline 458/488/514 nm (25 mW), and HeNe 561 nm (1 mW) and ×63 oil-immersion objective (Zeiss, NA 1.4) set to 37 °C. Cells were imaged by scanning the laser over a 13.5 × 13.5 μm area with the scan speed set to 8 or 16 and a digital zoom of ×10 or ×20, respectively. The diameter of the pinhole was 0.83 µm. Bleaching of the set region of interest (ROI) was performed on 20 × 50 or 30 × 80 pixel area (for ×10 or ×20 zoom, respectively) using 15 scan iterations and the corresponding laser power set to 100%. Two images were acquired before bleaching and ten images, with a time interval of 1 min, recorded after bleaching using an automatic time-course function. Instrument autofocus was used between images in reflection mode. All images were acquired using 2% maximum laser power. For all fluorescent images, corresponding differential interference contrast (DIC) images were recorded using transmitted light. All images were recorded using Zeiss Zen 2011 software.