Table 2.

Settings for imaging polarity establishment

Widefield	Channel	Transmitted light	EM gain	Exposure (ms)
	DIC	100 %	150	50
	GFP (470 nm)	2 %	750	250
	RFP (560 nm)	2 %	750	250
Spinning disk	Channel	Laser power	EM gain	Exposure (ms)
	DIC	N/A	200	100
	488 nm	10 %	200	200
	561 nm	10 %	200	200

If using one fluorescence channel, use a 30 z-plane stack with images 0.24 µm apart (see Note 10). If using two channels, use a 15 z-plane stack with images 0.5 µm apart. Image at 45 s intervals for 45–90 min total (see Subheading 2.5 for more details)