Zeiss 710 MP microscope
This page shows focus drift issues on the Zeiss 710 MP. There is a problem with ambient temperature, but the incubator holds focus well when heated above ambient.
The first test was an overnight timelapse at room temperature from approximately 4:00 PM on Jan 4 to 8:00 AM on Jan 5, 2011. This was done with the 63X N.A. 1.4 oil immersion objective with a sample of fluorescent tape mounted in water under a 1.5 coverslip and each Z stack was at 15 minute intervals. The sample drifted approximately 8 um in the Z axis and then held steady. The problem with the intensity falling off is due to the laser. To save the life of the laser, we tried using the laser at low power. This, obviously, was unstable. The following movie and montage of the movie are the sample shown as XZ projections in Imaris. Movie here.
The second test was an overnight timelapse with the incubator set at 39 degrees C and had two hours to equilibrate before beginning the overnight timelapse at approximately 4:00 PM on Jan 5. This shows very slight drift in the first hour, stability over night, and then large drift when the incubator was turned off. Movie here.
Conclusion:
For focus stability, incubator needs 2 to 3 hours warm up time. After you put you sample in, it will take an hour to become fully stable, but should not drift more than 1 or 2 um during this hour. Make the Z series for long time lapse begin and end with a margin of 1 to 2 um.
comments, questions, suggestions: Michael.Cammer@med.nyu.edu