For time-lapse recordings, images were captured every 3–5 min for a total of 8 h using Plan Fluor 10×/0.30 DIC L/N1 objectives with DM5500 and DMRXA2 compound microscopes (Leica Biosystems) at 18°C with either a DFC 300FX camera (Leica Biosystems) and LAS acquisition software or an Orca-5G camera (Hamamatsu Photonics) and SimplePCI software. For in vivo imaging, embryos were immobilized onto plasticine. Time-lapse and NCC tracking was performed using the ImageJ Manual Tracking plug-in as previously described (Carmona-Fontaine et al., 2008 (link); Matthews et al., 2008 (link)). Time-lapse imaging for CIL and CoA assays was performed at 18°C in Danilchick’s medium using an upright microscope (Eclipse 80i; Nikon) fitted with an objective (Plan Fluor 10×/0.30 DIC L/N1) and a camera (ORCA-05G; Hamamatsu Photonics). Data were acquired using SimplePCI software. Confocal images were acquired at 22°C in Danilchick’s medium using a TCS SPE upright microscope (Leica Biosystems) fitted with a HC PL APO 20×/0.75 IMM CS2 water objective. ISH images were captured at 18°C using a stereomicroscope (MZ FLIII; Leica Biosystems) fitted with a Plan 1.0×/0.125 objective and a camera (DFC420; Leica Biosystems). Data were acquired using IM50 v5 software (Leica Biosystems).
Protocol detail
Time-lapse Imaging of Cell Behaviors
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Apo a
Assays
Compound microscopes
Embryos
Microscope
Orca
Plasticine
Corresponding Organization : University College London
Other organizations : University of Messina, University of Parma
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Variable analysis
independent variables
- Time of image capture (every 3–5 min)
- Time-lapse recordings
- NCC tracking
- Objectives (Plan Fluor 10×/0.30 DIC L/N1)
- Microscopes (DM5500 and DMRXA2 compound microscopes from Leica Biosystems)
- Temperature (18°C)
- Cameras (DFC 300FX and Orca-5G)
- Acquisition software (LAS and SimplePCI)
- Embryo immobilization (onto plasticine)
- Imaging medium (Danilchick's medium)
- Microscope setup (upright Eclipse 80i; Nikon)
- Confocal microscope (TCS SPE upright microscope; Leica Biosystems)
- Stereomicroscope (MZ FLIII; Leica Biosystems)
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