We mounted embryos as described previously (Robin et al., 2014 (link)) on glass slides under #1.5 coverslips in 3–5 µl of standard Egg Salts containing ∼100 uniformly sized polystyrene beads (18.7 ± 0.03 µm diameter; no. NT29N; Bangs Laboratories). The beads acted as spacers and allowed us to achieve uniform compression of the embryo surface across experiments (Robin et al., 2014 (link)).
We performed all imaging at 21–23°C on a Nikon ECLIPSE-Ti inverted microscope equipped with a Ti-ND6-PFS Perfect Focus Unit. A laser merge module (Spectral Applied Research) controlled fast, tunable delivery of 481-nm and 561-nm laser excitation from 50 mW solid-state lasers (Coherent Technology) to a motorized TIRF illuminator. We adjusted the laser illumination angle to achieve near-TIRF illumination (Tokunaga et al., 2008 (link)). We collected images using a Nikon CFI Apo 1.45 NA oil immersion TIRF objective combined with 1.5× intermediate magnification onto an Andor iXon3 897 EMCCD camera. All image acquisition was controlled by using Metamorph software. We used ImageJ to set minimum and maximum pixel values and perform gamma adjustments on the original 16-bit image data before converting images to 8-bit red, green, blue format or grayscale format for display. We performed these operations identically for all images that are compared directly.
We performed all imaging at 21–23°C on a Nikon ECLIPSE-Ti inverted microscope equipped with a Ti-ND6-PFS Perfect Focus Unit. A laser merge module (Spectral Applied Research) controlled fast, tunable delivery of 481-nm and 561-nm laser excitation from 50 mW solid-state lasers (Coherent Technology) to a motorized TIRF illuminator. We adjusted the laser illumination angle to achieve near-TIRF illumination (Tokunaga et al., 2008 (link)). We collected images using a Nikon CFI Apo 1.45 NA oil immersion TIRF objective combined with 1.5× intermediate magnification onto an Andor iXon3 897 EMCCD camera. All image acquisition was controlled by using Metamorph software. We used ImageJ to set minimum and maximum pixel values and perform gamma adjustments on the original 16-bit image data before converting images to 8-bit red, green, blue format or grayscale format for display. We performed these operations identically for all images that are compared directly.
