Background correction for fluorescent modality

Use this procedure for step 6 of the slide scanning workflow.

Procedure

This procedure requires Chroma background correction slides. There are Chroma blank slides for each channel; these slides provide a uniform illumination and their intensity is even throughout the field of view. You can purchase those slides through Chroma or MBF Bioscience.

1. Focus on your tissue so that you can get close enough to the actual focal plane you'll be using.
2. Check clip detection.
3. Click the Acquire Background Correction Image button.
4. Remove the tissue slide from the stage and place the Chroma slide for the first channel on the stage.
5. Because the live image is probably very bright or red (because saturated/completely white pixels are shown in red when clip detection is turned on), decrease the exposure time with the slider and/or use the remote control to turn down your fluorescent light source's intensity until only a few scattered red pixels remain visible.
6. Click OK to acquire a background correction image for this channel.
7. Remove the Chroma slide for the first channel, and place the Chroma slide for the second channel on the stage, then repeat steps e-g for each subsequent channel.
8. Place your tissue slide on the stage again. It should still be in focus as a result of step a; if not, focus again.

   After you have acquired your background correction image, adjust the exposure time and histogram settings for each channel – for best practices, refer to this tutorial video: Fluorescence imaging

9. Proceed to the next step in the workflow.

When using background correction, we recommend that you acquire a sample image at step 8 of the workflow. If you're not satisfied with the sample image, refer to Slide scanning: Troubleshooting for fluorescence.