Instructions
5
Troubleshooting
5.1
Focus not Stable
Focus drift is a disturbing effect, especially during time-lapse experiments. 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 20 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.
5.2
Evaporation is Too High
Depending on the incubating conditions, small volumes might evaporate quickly, especially during
long–term experiments. We suggest using silicone oil (e.g. Anti-Evaporation Oil, ibidi, 50051) to
decrease evaporation. If you have an actively controlled humidifying device, increase the set value
for relative humidity.
5.2.1
Silicone Oil Procedure (Anti-Evaporation Oil)
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 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 by pressing the pipette tip directly on the upper side
of the reservoir. For example, when using the µ–Slide VI
0.4
, fill each reservoir with 30 µl cell–free
medium and 30 µl of silicone oil.
1
In case the experiment needs to be started immediately, either after placing the sample on the microscope or after
closing the lid, we recommend controlling the focus for 20 minutes. In the first minutes after starting the experiment,
temperature equilibration might influence the focus/z-position of the cells.
28
Heating System
Version (26th April 2017)
