When you want to take images of positions/ tile regions on a sample carrier, that has to be taken off the stage, e.g. for incubation purposes or changes of the immersion medium, proceed as follows to re-position your sample carrier.

Starting the Experiment

  1. You have run the stage calibration and have located your sample, see chapter Calibrating the Stage and Selecting the Channel.
  2. You have set up at least one channel and adjusted the light/ camera exposure time.
  3. You have activated the Tiles checkbox and the Show All mode
  1. In the Tiles tool, open the Sample Carrier section and click on Select.
  2. The Select Template dialog opens.
  3. Select the template of your choice.
  4. Adjust the surface of the sample carrier. Refer to the chapter Creating a Global Focus Surface.
  5. Click on OK.
  6. The Select Template dialog closes.
  7. Click on Calibrate.
  8. The Sample Carrier Calibration Wizard opens.
  9. Move a sample reference point into the middle of the crosshair. This reference point can be any unique, identifiable point on the slide and does not have to in the middle of the slide.
  10. Click on Next.
  11. Under X/Y Position click on Set Zero.
  12. Click on Next.
  13. As Calibration Method, select Search Reference Point
  14. Click on Next.
  15. Click on Set Current X/Y.
  16. In the Tiles tool, click on Show Viewer and add positions/tile regions at your locations of interest.
  17. Once you have defined all your positions/ tile regions, click on Options on the Acquisition tab to in the Experiment Manager.
  18. Save your experimental settings, including the lists of positions/ tile regions, by selecting either the Save As or Export entry.
  19. Start your experiment and record images from your selected positions/ tile regions.
  20. Remove your sample off the stage and e.g. put it back into the incubation chamber.
  21. Close the software.
  1. You have done all settings for a successful re-positioning of your sample carrier after the experiment.

  1. For demo purposes, select a standard slide that can mimic your test sample.
  2. Regarding calibration of your template, you can customize your own carrier see the chapter Customizing a Sample Carrier Template, but for slides with one coverslip or well, there is only the option for Single Reference Point Calibration. For Multi-well plates, you will have the option for 7-point, 4-point, 3-point or 1-point calibration. This becomes important for adjusting for the rotation of the sample.
  3. It is assumed that you just use a conventional glass slide with some cells or tissue that is positioned in the center.
  4. You can zoom in and out using the mouse scroller, and move the stage in the Center Screen Area to a point of interest with a double-click on the sample carrier.
  5. With Save As the settings will be saved directly in the Experiment Manager. With Export the settings will be saved in a folder of your choice.

Re-Positioning of the Sample Carrier after the experiment

If you cycle the power on the microscope the software will prompt you, to calibrate the stage and/ or focus drives. Thus, if the calibration of the multi-well plate was performed under the same conditions, then the sample carrier calibration will still be valid. You must however ensure that other parameters like plate orientation and placement on the microscope have not changed.

  1. Restart the software.
  2. In the Acquisition tab go to the Experiment Manager and Reload or Import your experimental settings including your list of positions/ tile regions.
  3. In Tiles tool under Sample Carrier, click on Calibrate.
  4. The Sample Carrier Calibration Wizard opens.
  5. Move your previously chosen sample reference point into the middle of the crosshair.
  6. Click on Next.
  7. Under X/Y Position click on Set Zero.
  8. Click on Next.
  9. As Calibration Method, select Search Reference Point.
  10. Click on Next.
  11. Click on Set Current X/Y.
  12. Now, you still need to verify the Z-offset of your positions. Therefore, follow the corresponding instructions given in the chapters Adjusting Z-Values of Tile Regions and Adjusting Z-Values of Positions.
  1. All of your selected positions/tile regions are now re-assigned to the correct X/Y/Z-values in relation to your (unique identifiable) reference point.
  2. You can re-start your experiment and record images from your selected positions/tile regions.