Eggs were obtained by dissolving gravid hermaphrodites in a solution of sodium hydroxide and sodium hypochlorite followed by three washes with M9. Drops of M9 containing eggs were plated onto nematode growth medium plates seeded with Escherichia coli OP50, and incubated at 20°C for 68 h. Animals close to their first ovulation were selected for imaging, anaesthetized with 0.01% tetramisole and 0.1% tricaine in M9 buffer, and mounted on 2% agarose pads.
Images were acquired on a Nikon Eclipse 80i microscope using a 60× 1.40-NA objective. Fluorescence was monitored using a standard GFP filter set (excitation 470/40, beam splitter 495, emission 525/50; Chroma Technology), with fluorescence excitation provided by a Nikon Intensilight C-HGFI 130-W mercury lamp, attenuated at the source with the ND16 neutral density filter, and shuttered between exposures using a SmartShutter (Sutter Instruments) triggered by the camera transistor-transistor logic signal.
Images were captured using a SPOT RT3 monochrome CCD camera, and acquisitions were controlled by SPOT Advanced 5 software (Diagnostic Instruments). Images were acquired at a rate of one frame per second, an exposure time of 75 ms, and a gain of 8. The full chip of the camera was used, generating images 1,600 × 1,200 pixels in size. Time-lapse image sequences were saved as 8-bit TIF files.
Images were acquired on a Nikon Eclipse 80i microscope using a 60× 1.40-NA objective. Fluorescence was monitored using a standard GFP filter set (excitation 470/40, beam splitter 495, emission 525/50; Chroma Technology), with fluorescence excitation provided by a Nikon Intensilight C-HGFI 130-W mercury lamp, attenuated at the source with the ND16 neutral density filter, and shuttered between exposures using a SmartShutter (Sutter Instruments) triggered by the camera transistor-transistor logic signal.
Images were captured using a SPOT RT3 monochrome CCD camera, and acquisitions were controlled by SPOT Advanced 5 software (Diagnostic Instruments). Images were acquired at a rate of one frame per second, an exposure time of 75 ms, and a gain of 8. The full chip of the camera was used, generating images 1,600 × 1,200 pixels in size. Time-lapse image sequences were saved as 8-bit TIF files.