TABLE 2.

Troubleshooting table

Step	Problem	Possible reason	Solution
9	Too many bubbles around worms	Too little anesthetic liquid left on slide	Stop removing anesthetic with the mouth pipette once the liquid no longer easily fills the capillary glass without applying suction
	Worms too widely distributed on the agarose pad	Too much anesthetic liquid is left on the pad	Try to minimize the amount of liquid transferred with the worms by gently tapping the glass spot plate (the worms will coalesce at the bottom of the well (see Fig. 3c), allowing you to draw them up in a minimal amount of liquid)
10	Worms become sick or die during imaging	Too much OP50 is transferred with worms Worms are injured while they are anesthetized, transferred, and covered by a coverslip The interface between the coverslip and the agarose pad is not properly sealed	Use a minimal amount of bacteria to transfer worms Use care when handling worms at all points Use a small paintbrush to apply VALAP to four sides of the coverslip and leave a small gap for gas exchange
14	Worms are not at the correct stage in development to image	Wrong sized animals are picked Worms cultured for too short or long after syncing Worms not kept at an optimal temperature	Sync animals by bleaching instead of trying to pick from mixed aged plates Optimize the correct incubation time and temperature for each strain
15	Specimens with low fluorescence signal will result in grainy images	EM Gain might be too high while exposure time is likely too short	Adjust the settings until high-quality images are acquired
	Worms start moving during time-lapse	Worms are too crowded or the incubation in anesthetic was too short	Lengthen incubation time in anesthetic, avoid mounted worms that are touching each other, as they tend to move more during time-lapses
	Photobleaching occurs during imaging	Worms are exposed to too much cumulative laser power	Reduce laser power, exposure time
17	Imaging data cannot be loaded into Imaris	The format of data files is not properly selected in Imaris	Try to correctly match file format selected in Imaris with that of raw data files
18	Disproportionate 3D rendering	Geometry set in Imaris does not match acquired raw images	Verify correct voxel dimensions. If voxel size was determined by calculation, confirm with a stage micrometer