Table 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
11	Cells are motile	Too much liquid on the pad surface before transferring it to the imaging surface	Wait until the pad surface dries completely after seeding pads
		Damage to agarose pad	Avoid marring the agarose surface with the pipette tip when seeding bacteria, or with the scalpel when transferring pads to imaging surface
12	Cells are not fluorescent	Reporter not optimal	If necessary, optimize the reporter ribosome-binding site or codon usage. Check a constitutive promoter if you are working with a new fluorescent protein for the first time
		Low-expressing reporter	If possible, use an alternative reporter or increase the reporter copy number by using a plasmid-based system or by using reporter repeats separated by ribosome-binding sites
16	Cells do not grow, even outside of the acquisition area (field of view)	Old medium	Remake cell medium components from fresh stocks
		Cell density may have a role in normal growth of some bacteria	Vary the pad cell densities and check if growth improves
		Antibiotic concentration is too high	Unless necessary, we often omit antibiotics in the pad entirely. When this is unavoidable, a decrease in antibiotic concentration may be prudent, especially with minimal defined medium
	Cells grow healthily outside the field of view but grow poorly inside it	Phototoxicity	Reduce the frame frequency or reduce the amount of excitation light
	The microcolony shifts very quickly during the course of the movie	Agarose pad evaporation	Ensure that the imaging sample is sealed with either sealing grease or Parafilm
	The microcolony shifts gradually during the course of the movie	Slight agarose pad size changes	Some amount of microcolony shift is expected, and it can be minimized by using larger agarose pads and imaging closer to the center of the agarose. A microcolony anti-shifting routine can also be enabled during acquisition
	The movie loses focus	Within the first few frames, initial focus drift may exceed the autofocus range	Before starting acquisition, ensure that focus drift is minimized. If this does not fix the problem, increase the autofocus range
		If focus is poor throughout the movie, the phase (bright-field) image may be blurry, causing the software autofocus to fail	Ensure a smooth agarose surface for imaging and avoid cells near the edge of the pad where the phase image is not ideal
		Insufficient immersion oil on the underside of imaging dish	Spread enough oil evenly among the pads being imaged