Sp8 inverted microscope

Embryos were anaesthetized in 0.01% tricaine and pre-selected based on the expression of the desired fluorophore using a Nikon ZMZ18 fluorescent stereoscope. Embryos were embedded in 1–1.2% low-melting (LM) agarose (PeqGOLD Low Melt Agarose, PeqLab Biotechnologie GmbH), dissolved in E3 medium with 0.01% tricaine. Embryos were mounted on glass-bottom dishes (Greiner Bio-One #627871) for confocal microscopy or pulled together with agar into glass capillaries (Brand, #701904) with a rod (Brand, #701932), and then pushed halfway out, into the microscopy chamber for light-sheet microscopy. Both the imaging dishes and the microscopy chamber were filled with E3 medium containing 0.002% PTU and 0.01% tricaine during the entire imaging period. For confocal microscopy in Figure 6—figure supplement 1, Leica SP8 Inverted microscope with a Leica ×40/NA 1.1 WI objective was used to capture 75-μm-thick stacks with z-step of 1 μm. For all other confocal microscopy, an Andor Dragonfly 200 Sona spinning-disc microscope with a Nikon ×20/NA 0.95 WI objective and 40 μm spinning disc was used to capture imaging 70–75-μm-thick stacks with z-steps of 2 μm, every 60 s. For light-sheet microscopy, a Zeiss Z.1 microscope with a W Plan-APO ×20/NA 1.0 WI imaging objective and ×10/NA 0.2 air illumination objectives was used to capture 40–75-μm-thick stacks with a z-step of 0.5–0.75 μm, every 15–30 s.

To prepare cells for photobleaching experiments, WT and PLK4 KO mTECs expressing GFP-Centrin were isolated and differentiated as described above. On ALI day 3, the filters with adhered cells were cut out from the transwell insert and inverted onto a glass coverslip with a drop of differentiation media. A plastic ring was placed on top of the filter to keep it in contact with the coverslip. Cells were imaged on a Leica Sp8 inverted microscope using a 63×/1.4 NA objective. An region of interest (ROI) was drawn around all or part of a GFP-Centrin signal at the centrioles or aggregates and photobleached to approximately 80% of the pre-bleach fluorescence intensity with 10 pulses of 488 nm laser light (1039 ms exposure). A Z-series of 13 planes (0.5 μm step size) was acquired immediately before and after bleaching. GFP-Centrin fluorescence intensities within the bleached ROI were measured using ImageJ. The first post-bleach intensity was subtracted from the pre-bleach and post-bleach values and then normalized to give the percentage of the pre-bleach intensity.