timeseries, large images and focus

Posted by Sarah Chacko on
URL: http://confocal-microscopy-list.275.s1.nabble.com/timeseries-large-images-and-focus-tp5747682.html

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Hello,

I am using a Nikon A1 confocal and I am trying to take a time series over several hours,  scanning across channels in a microfluidic chamber - my channels are 20 fields of view across, there are three channels.

I am finding that even with one long (20 fields of view) image across one channel the focus is lost half way across, and moving to another region nearly always loses focus.

I could increase the pinhole or take a small z stack so that being out of focus would matter less - I'm looking at bacteria ~1 micron in size, but don't need to resolve anything smaller, just distinguish between them using fluorescent labels.

I wondered whether in principle this should work, using long image and PFS with several starting points, and if anyone has something similar working well, or whether I am expecting too much.

Sarah
(student)