Gas Incubation System
8
Troubleshooting
8.1
Focus not Stable
Focus drift is detrimental for most microscopy experiments, especially long duration time-lapse ex-
periments. Focus stability is mainly influenced by mechanical changes and temperature variations.
Follow these recommendations to keep your cells in focus:
• Switch on all components (e.g., heating, gas incubation, computer, or other equipment) at least
60 minutes before starting the experiment.
• After you put the sample onto the microscope, wait 30 minutes before starting a time-lapse
experiment to achieve temperature and immersion oil equilibration
• Keep the room temperature as stable as possible. Air conditioning should either be working
continuously or switched off.
• Do not change the temperature during the experiments. Avoid door/window openings, as this
could rapidly change the temperature.
• Eliminate all sources of mechanical vibrations. Use a damped table for your microscope.
8.2
Evaporation Is too High
Depending on the incubating conditions, small volumes might evaporate quickly, especially during
long-term experiments. If you have an actively controlled humidifying device (e.g. ibidi Gas Incuba-
tion System), increase the set value for relative humidity. Additionally, we suggest using silicone oil
(e.g. Anti-Evaporation Oil, ibidi, 50051) to decrease evaporation.
Covering the medium with sterile silicone oil prevents all evaporation effects and is compatible with
cell culture. Please do not use mineral oil, as this can be harmful to your cultureware.
Equilibrate oil and medium inside the incubator overnight. This step helps to avoid the formation of
air bubbles, and pre-warms the solutions to 37°C. Afterwards, fill your slide with cells and medium.
Cover the medium’s surface with an appropriate amount of silicone oil. Don’t drip the oil directly
onto the surface, but let it run down the edges of the cell culture vessel. Details about avoiding
evaporation are given on the ibidi web site in
Application Note 12 ”Avoiding Evaporation”
8.3
Gas Flow too Low
8.3.1
Gas Input Open Without Connected Gas
Always connect all gas input ports with the appropriate gas and pressure (1 bar/14.5 psi). Do not
leave any input ports open. In case N
2
is not needed or available, close the input port by using the
provided dead-end tubing.
2
If the experiment needs to be started immediately, either after placing the sample on the microscope or after closing the
lid, we recommend checking the focus for 20 minutes. In the first few minutes after starting the experiment, temperature
equilibration might influence the focus/z-position of the cells.
Instruction Manual
33
Version 2.2 (May 15, 2019)
