Channel Mapping Calibration

Channel Mapping Calibration

Channel Mapping Calibration is a complex non-linear calibration process, which is required for multi-color imaging (e.g. co-localization).

Calibration Instructions

  1. Connect to the microscope, start the temperature control, and wait until the temperature becomes stable before moving forward.
  2. Place a bead slide that is visible in both channels (e.g. TetraSpeck microspheres) on the microscope with fresh oil. The beads should be non-clustered, homogeneously spread in the field of view, and a couple of hundreds of them are ideal for calibration. For more instructions on how to prepare a bead slide, check here​.
  3. Click on View to start the camera, find the focus of the bead slide using any laser, set the focus reference, and enable the Z lock. For more details on how to set the focus reference, check here.
  4. Change the contrast method to Photon Count, and turn on lasers with enough power to render the beads with comparable intensities in both channels at 30 ms exposure. For example, use both green and red lasers to adjust the channel intensities. 
  5. Click on Instrument > Channel Mapping Calibration from the toolbar to open the calibration window.
  6. In the pop-up window, click on Calculate Mapping to start calibration with default parameters. In some systems, there are several parameters that can be tuned for calibration.

Acceptance Criteria

  1. Evaluate the calibration results and click on Save Mapping to save the calibration result to a "c_map.nim" file if:
    1. Standard Deviation of Errors must be less than 20 nm (preferably less than 15 nm).
    2. Point Coverage is 100%.
    3. Mapping Quality is good.

  2. In case of a poor calibration, either click on Reset Mapping To Default to retrieve the default mapping or move the slide to another area and rerun the calibration (trials and errors). If the calibration fails in multiple areas, consider switching to another bead slide. If the calibration fails with multiple samples, contact the CX team for help.

Discussions

  1. Channel mapping calibration MUST be carried out after the microscope reaches its working temperature. You MUST rerun the calibration if the imaging temperature has been changed.
  2. It is recommended to run the channel mapping calibration at least once per day. You can change the display method to Color Overlay to assess the channel alignment.
  3. The Z lock MUST be enabled before running the calibration.
  4. In the case of using only one channel for imaging, channel mapping is not required.
  5. For 3D Channel Mapping Calibration, click on Engage 3D Lens before running the calibration. The 2D and 3D calibration results will be saved separately for NimOS 1.19.1 or later versions. For instructions on how to upgrade NimOS, check here. 

If at any point you are having issues with the calibration process, please do not hesitate to contact the CX team.


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