Lsm 510 live confocal microscope

Six-somite stage embryos Tg(actb2:Mmu.Arl13b-GFP) were embedded in 1% low-temperature melting agarose (Biozym) in 30% Danieau’s solution (17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO₄ × 7 H₂O, 0.18 mM Ca[NO₃]₂, 1.5 mM HEPES). Real-time imaging of GFP-labeled KV cilia was performed on a ZEISS LSM 510 Live confocal microscope equipped with a LD LCI Plan-Apochromat 25×/0.8 glycerine objective (Zeiss). Six hundred images were recorded per sample at 9–10 frames per second with a resolution of 512 × 512 pixels. Similarly, flow in the KV of embedded 8-somite stage embryos was visualized by particle tracking using differential interference contrast (DIC) microscopy (Zeiss Axio Observer microscope; Zeiss) for 2 min at 20 frames per second. [39 (link)]. For high-resolution 3D confocal imaging of zebrafish KV, Tg(actb2:Mmu.Arl13b-GFP) was fixed in methanol and stained anti-GFP to enhance contrast as described previously [83 (link)]. A Leica TCS SP8 STED 3X microscope was used for image acquisition. Vertical projections of recorded stacks were generated using ImageJ 2.

Zeiss LSM 510 live confocal microscope was used for imaging cpYFP fluorescence. Images were captured with a ×40, 1.2 NA water immersion objective at a sampling rate of 1 s/frame. Dual excitation of mt-cpYFP was achieved by alternating excitation at 405 and 488 nm, and the emission was collected at >505 nm. Experiments were performed at room temperature (23 °C). Time-lapse confocal images were analyzed using custom-developed algorithms written in Interactive Data language (IDL) [28 (link)] and ImageJ (NIH). Motion artifacts, background subtraction and photo-bleach correction were taken into account before automatic flash detection, using the built in custom-software.