Live Cell Tracking

Important

Take images in as few fields of view as possible, with as few conditions as possible. Live tracking generates large quantities of data.

  1. At least 1 hour before imaging, turn on CO2 tank. Turn top knob to be completely open, and open valve so that it is inline with the tubing. Left gauge should read ~15-20 psi, right should read over 600 psi.

  2. Also at this time, turn on the Okolab controller display (E) so that it can start getting up to temp.

  3. Follow directions for startup.

  4. Ensure water tank is full to max line. Refill with sterile water and a serological.

  5. Replace stage with incubator stage (completely enclosed) if necessary.

  6. Turn on stage sensing (often the empty plates by the computer are better for sensing).

  7. Use the experiment suitable to your fluorescent needs (under the Fluorescence tab on the right panel).

  8. Find PFS offset (under PFS tab in rightmost panel).

  9. Click on Job Wizard (in the bottom left corner).

  10. If using an experiment from a previous run, double click to continue editing. If you click run and edited anything, you must save it under a new name.

  11. Turn on autofocus on brightfield (or whatever channel is constant between all conditions).

  12. Autofocus on one point.

  13. Turn on use autofocus when PFS fails.

  14. Do a test run (even if using a previous template, you must click on each module before it will let you proceed).

  15. Let the plate sit for ~20 min before starting image collection.

  16. To do media changes, hit the pause button in the corner (hit resume when plate is back in place).

  17. When live track is completed, follow directions for shutdown.

Note

MC has noticed the glass bottom plates get external gunk more easily than the plastic. If you’re noticing this in your BF, clean off all surfaces with a kim wipe and EtOH.