TABLE 3.

Troubleshooting table.

Step	Problem	Possible reason	Solution
15	No environmental chamber available		Carry out all steps after Step 18 at room temperature
20	Difficulty focusing on cells	Small size of E. coli cells or low cell density	Focus on cells expressing Spinach using fluorescence in the FITC channel
		Poor magnification of microscope	Make sure the miscroscope in use has an objective capable of imaging E. coli (i.e., ×60–×100 with >1.4 NA)
20	No adhered cells	Residual LB medium in samples can inhibit cell adhesion	Wash cells in M9 media twice before adding them to wells (Step 24)
		Incomplete poly-lysination	Check freshness and concentration of PLL stock. Carry out second PLL coating (Step 7)
22	No fluorescence signal	Improper concentration of DFHBI	Check stock solutions and remake imaging medium
		No Spinach expression	Check for Spinach expression by reverse transcription (RT)-PCR or by other means
23	Cannot save and move between x, y and z positions	No automated stage	Carry out time-course measurements on a per-sample basis
29	Greater than 1.5-fold changes in Spinach signal over time	Incorrect pH of media	Check that all media solutions were made properly and that the pH is adjusted to 6.0