For the acquisition and subsequent preparation of reference scans, note the following:

• All reference slides need to be scanned. It is not mandatory to scan whole slides, but it is recommended to reduce imaging loops and optimize processing.
• All slides need to be scanned with exactly the same channels (the same scan profile) as the later multiplex slides. The only exception is the exposure time. For acquiring the reference scans, the exposure times may be different from the exposure times used for the multiplex scans later.
• We recommend, however, to set the exposure times for the reference scans either identical to the intended exposure times of the multiplex scans or multiplied by a constant factor for all channels (e.g. 2x).
• We recommend using a factor of 2x or 3x, if necessary, to use the full camera dynamic range but without over exposure. This will optimize the SNR in the reference scans and can result in more robust unmixing results later.
• The optimal quality of the refence scans is crucial for building a precise and robust unmixing library. For quality control, open all reference scans and set the histogram the same for all scans such that in each scan the dye is clearly visible (except for the unstained tissue).
• Good single-stained slide reference scans have a clearly visible and specific signal in the intended channel. Signals in other channels are either crosstalk or autofluorescence. The unstained reference sample may only contain autofluorescence.
• The tissue can contain different types of autofluorescence which can be best seen in the unstained reference sample scan. As an example, there can be autofluorescence portions of small, bright spots or clusters, resulting from red blood cells, medium bright vessel-like structures, and dimmer structures in the major part of the tissue. Different types of autofluorescence typically have (slightly) different spectral properties. Hence, the unmixing is always optimal for one particular type of autofluorescence and not perfect for others.
• For the reference spectra creation, it is important to decide for one autofluorescence type which co-locates most with specific signals.
• Use the function Create Image Subset on the Processing tab to select and crop a small part of each reference scan which best fulfills the following conditions:
• The specific signal of the single-stain is clearly expressed in this area.
• The type of autofluorescence selected above is available at least partly in this area.
• Optimally, the cropped area contains a few camera frames (tiles). In order to ensure that at least one full tile is available in the cropped image, use the Keep Tiles option of the function.
• If in doubt, create multiple crops from the same images. The selection of the tiles for the spectra creation is the most important parameter which can be optimized to fine-tune the quality of the resulting unmixing.
• Save all cropped images to a folder.