W plan apochromat 20x 1.0 dic objective

C8161 cells were dual labeled with H2B:mCherry and H2B:PSCFP2 (photo-switchable cyan fluorescent protein) by lentiviral infection. Cells were transplanted into the chick embryo neural tube as described above. A Teflon membrane window was installed in the egg as previously described (Kulesa et al., 2010a). H2B:PSCFP2-labeled cells were photoconverted within 1 hour following transplantation. Photoconversion was performed on a Zeiss 710 multi-photon upright microscope using 2-photon excitation at 780nm and a W Plan-Apochromat 20x/1.0 DIC objective (Zeiss).

Individual embryos were removed from eggs with paper rings, rinsed with Ringer’s solution and placed dorsal side up within a thin ring of high-vacuum grease (Dow Corning) on 22×75mm microslides. Embryos were imaged using a Zeiss 710 multi-photon upright microscope. 2-photon z-stacks of dual labeled C8161 Gap43:CFP::H2B:YFP were acquired at a wavelength of 850nm. RFP-labeled EphB6+ C8161 cells were imaged with an excitation of 561nm. Images using a W Plan-Apochromat 20x/1.0 DIC objective (Zeiss).

The time-lapse imaging platform, including sample preparation, is detailed in a previously published protocol (Kulesa et al., 2010a). Briefly, Teflon windows were were placed over the developing chick embryo and sealed with beeswax. The egg was maintained in an environmental box placed around a Zeiss 710 multi-photon upright microscope stage and heated to 38°C. The chamber was humidified with a sponge placed in a dish of water. Non-evaporating immersion liquid (Immersol W, Carl Zeiss) was used to bridge the W Plan-Apochromat 20x/1.0 DIC objective (Zeiss) with the Teflon membrane. Simultaneous 2-photon excitation of the dual labeled C8161 Gap43:CFP::H2B:YFP cells was achieved at a wavelength of 850nm. Z-stacks with a 10um z-slice were acquired every 7 minutes for 18 hours.