7. Background correction

1. As instructed onscreen, navigate to an area on the slide where there is no tissue or debris, but the coverslip and mounting medium (if present) are in the field of view.

   Check the box for Joy Track. Note that this option will be grayed out if Joy Track is already enabled.

2. Check the box to turn on Clip Detection. This will identify overexposed areas in the field of view with dark red pixels.

3. Adjust the exposure (in Camera Settings) so that only a few scattered red pixels appear in the white areas (the image should be very bright but not saturated). Typically, keep the exposure between 10 and 100 ms.

4. Click the white balance button to obtain automatic white balance.

   As an alternative on systems with a color camera, you can manually set the white balance by clicking the camera settings button; find instructions here.

5. Click the Acquire Background Correction image button.

   The background image is acquired and displayed onscreen; also it is listed in the image organizer.

   • Ensure that the background image appears completely white and free of debris.
   • If there is debris, adjust the field of view to avoid the debris and acquire the image again.

   Note that the most recent background image captured is used to correct background in the acquisition.

Do not adjust the exposure time or light level once you have acquired your background image.

This procedure requires "autofluorescent plastic slides" for each color channel; they provide uniform light emission throughout the field of view. You can purchase these slides through either Chroma Technologies (Autofluorescent Plastic Slides) or MBF Bioscience.

1. Focus on your specimen so that you can acquire a background image at, or close to, the focal plane you will use for the slide scan.

2. Check the box for clip detection—saturated pixels will be shown in red.

3. Click the Acquire Background Correction Image button. You will be prompted to change slides and camera settings; here is what you can expect:

    

   • Remove the tissue slide from the stage and place the autofluorescent slide (Chroma Technologies) for the first channel on the stage.

   • Because Clip detection is enabled, saturated pixels will be shown in red (the entire window will likely be red). Adjust the exposure using the slider so that only a few scattered red pixels remain (the image should be very bright but not saturated).

   • Click OK to acquire a background correction image for this channel.

   • Following the onscreen instructions, remove the autofluorescent slide for the first channel, and place the slide for the second channel on the stage, then repeat the adjustments and click OK for each subsequent channel. Repeat if/as instructed onscreen.

4. Place your specimen slide on the stage again. It should still be in focus; if it is not, focus again.

5. Use the Multichannel Control panel to adjust the exposure time and camera histogram settings for each color channel you will use for the slide scan.

   For best practices, refer to this tutorial video: Fluorescence imaging.

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