Keyence Time Lapse Imaging

Setting up the incubation chamber

The Keyence comes with a special chamber / plate holder and stage top incubator controller for time lapses that maintains temperature, humidity, and CO2 levels for the cells.

  1. Place the stage adaptor in the Keyence and use a screwdriver to tighten the screws.

  2. Place the chamber on top of the stage adaptor. It just sits in place.

  3. Secure the cords/tubes to the plate using the clasps.

  4. Remove the chamber lid and add Elga water to the “moat” until it is full. You can do this using the syringe located by the Keyence. Be careful not to spill water into the Keyence!

  5. Turn on the stage top incubator controller that is attached to the incubation chamber.

  6. Turn on the flow of CO2 by opening the valves on the tank. There are 3 different values you may need to open.

  7. Check there is enough CO2 tank pressure (right pressure gauge) for your time lapse (should be >600 psi?). The left pressure gauge should be between 14.5-21.5 psi; to change this pressure you can turn the large blue valve on the CO2 tank. Ensure the CO2 is flowing by looking at the process value (PV) on the controller display.

Using the Keyence Software

  1. Open the Keyence software and select “Time Lapse Capture”.

  2. Set capture points, z-stacking, and stitching as normal.

  3. Click “Start Capture”.

  4. Choose where you want to save the images. You should save them onto the Galloway Lab OneDrive that is specifically for time lapses.

  5. Choose how frequently you want to capture images. They software will show the limit for how frequently you are able to capture images. This limit depends on the total number of images you want to capture at each time point.

  6. Choose how many times you want to capture images. The software will display the total capture time in the window based on imaging frequency and number of time points.

  7. There is also a limit to the total number of images you can take in a single time lapse (100,000 images). You might also be limited by how much storage space is available on the Keyence computer. You can free up space on the computer if you need more storage space up to ~1.8TB.

  8. Once all the capture conditions are set, you can begin the time lapse.

Tips and Tricks

  • Mix light cells (fluorescent) with a population of dark cells (no fluorescence). This is helpful for tracking since it reduces crowding of the light cells. The ratio of light to dark you want will depend on your experiment.

  • You can also try to seed cells more sparsely than usual to prevent crowding of cells which helps with tracking.

  • Imaging as frequently as possible is best for tracking so the cells move less between time points.

  • Free up space on the Keyence computer the morning before you start the time lapse. This can be done by right clicking on OneDrive folders and selecting “Free up space”. It can take several hours for OneDrive to remove files from the computer.

  • Let the plate sit in the Keyence for a while (1-2 hours) before starting the time lapse. This will help keep the cells in focus during the time lapse. If you start imaging immediately, the cells will likely go out of focus within the first few hours.

  • Use a z-stack to ensure you keep the cells in focus throughout the time lapse. Brittany has previously used a z-stack with 60um range with a 12um pitch.