Table 2.

Troubleshooting for live imaging

Problem	Possible reason	Solutions
Migration does not complete	Phenotype of mutant	Confirm phenotype in fixed samples
	Ovary was dissected from a fly that was old or ill fattened	Use young and well-fattened flies
	Dissection damage	Dissect quickly and do not touch stage 9 with the forceps
	Medium is old or of wrong pH	Make new medium
	Surrounded by late-stage egg chambers or too crowded	Remove late-stage egg chambers and mount sparsely
	Compressed by cover slip	Leave enough medium for mounting
	Phototoxicity	Reduce laser power or scan time
Signal gets weaker later	Laser power too strong	Reduce laser power and open up pinhole
	Scanning time too long	Increase scan speed
Egg chamber drifts a lot	Excess medium	Remove excess medium so stage 9 moves slowly when the dish is tilted
	Big air bubbles	Carefully put on the cover slip to avoid big bubbles
	Excess halocarbon oil	Apply a thin layer of halocarbon oil
	Dish not loaded horizontally in the sample holder	Fit the dish well into the sample holder
	Stage moves too fast	Choose positions that are not too far away
	Stage 9 is close to germarium or ovariole has remaining muscle sheath	Avoid such egg chambers
	Ovariole is twisted and egg chamber moves when adjusting focus	Avoid such egg chambers
Egg chamber degenerates later	Dissection damage	Dissect quickly and do not touch stage 9 with sharp tip of the forceps
	Medium is old or of wrong pH	Make new medium
	Surrounded by late-stage egg chambers or too crowded	Remove late-stage egg chambers and mount sparsely