I’m still thinking part of it is that something about the PDMS is changing. Can you encase the PDMS in something impervious, like a small cap?
Have you repeated a small position time series on the PDMS-only sample? I can’t remember if you’ve already stated this, but be sure to adjust the delay/Interval so that the time difference between the first position for each round is similar to that difference when you’re running 500-1000 positions so that total time is duplicated.
When you ran it on the glass-air (not sample, no PDMS chamber) were you doing the full 500-1000 position time series?