1. You have started the wizard for calibrated unmixing, see Starting the Calibrated Unmixing Wizard.
2. You have prepared reference scans of your sample, see Information for Acquisition and Preparation of Reference Scans.

1. In the selection field, click .
2. A file browser opens.
2. Select the image of your unstained reference scan and click Open. If a spectrum of the autofluorescence is already available from a previous run of this wizard, select the spectra file.
3. The file is loaded. The image or the spectrum of the selected file is displayed in the Center Screen Area.
3. In case you have loaded an image, go to the Graphics tab below the image.
4. The controls for graphical elements are displayed, see Graphics Tab.
4. Use the 2D or Split view and the controls of the Graphics tab to draw at least one graphical element which contains a typical sample of the relevant type of autofluorescence.
5. You have marked the autofluorescence.
5. If a part of the image also contains only background, for example no tissue but empty glass, draw a graphical element into the image to mark the background.
6. The relevant information is marked in the image.
6. Go to Select Autofluorescence and select the graphical element that marks the autofluorescence.
7. Autofluorescence is set.
7. Go to Select Background and select the graphical element that marks the background, if available. Otherwise select None from the drop-down list.
8. The background is set.
8. Click Next.
9. The next step opens, see Selecting Dye Spectra.