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. 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 > 3D Mapping Calibration from the toolbar to initialize the calibration.
   
6. In the pop-up window, click on Begin Calibration to start the calibration.
   
   
7. Once the acquisition and model fitting is finished (which usually takes 5~10 minutes), a new window will appear to show the calibration results.
   
8. Click on Yes to accept the calibration if all values are within the acceptance criteria. Click on No to abandon the calibration if a value is outside the acceptance criteria. Note that a 3D channel mapping calibration will be run regardless of the selection, however, the fitting result will only be saved to the "3dmap.nim" file if accepted.

Acceptance Criteria

1. Evaluate the calibration results from the pop-up window and click on Yes to save the calibration to a "3dmap.nim" file if:
   
1. Beads Remaining after Filtering is more than 100.
   
2. Percentage of Beads Remaining after Filtering is more than 60%.
   
3. Percentage of the FOV Covered by Beads is more than 75%
   
4. Mean Error is less than 40 nm.

1. In case of a poor calibration shown above, click on No to abandon the calibration. 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.

Discussion

1. For video instructions on how to perform a 3D Mapping Calibration, check here.
   
2. In some systems, you will have to disengage and engage the 3D lens once to apply the calibration for 3D imaging.
   
3. Since the 3D Channel Mapping Calibration will be performed at the end of the 3D Mapping Calibration, it is recommended to perform the calibration after the microscope reaches its working temperature.

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