1. You have selected the autofluorescence spectra and are in the second step of the Calibrated Unmixing wizard, see Selecting Autofluorescence Spectra.
  2. You have prepared reference scans of your sample, see Information for Acquisition and Preparation of Reference Scans.
  1. For Unmixing Database Folder, define a name for the dye set to be used in the unmixing library.
  2. You have defined a name.
  3. Go to the table in the Center Screen Area, select the first entry, and click .
  4. A file browser opens.
  5. Select one of the (cropped) single-stain reference scan images and click Open.
  6. The file name is displayed in the table and the image is displayed below the table for the selected entry.
  7. For ChName, select the channel where the dye is most visible. Use the Split view of the image displayed below the table to identify the correct channel.
  8. You have selected the channel.
  9. For Dye, enter a name for the dye for later reference.
  10. Dye name is set.
  11. For AF, select the channel which mostly represents pure autofluorescence (i.e. has no specific signal) and has a good signal-to-noise ratio. Usually, the channels suggested by the system can be used. Otherwise, Use the Split view of the image displayed below the table to identify the correct channel.
  12. The channel for autofluorescence is set.
  13. Repeat the previous steps with the single-stain reference scans for all dyes, one per row.
  14. The dye spectra are set.
  15. Click Next.
  16. The next step opens, see Checking the Calculated Dye Spectra.