Axioscan Advanced Spectral Unmixing

This module provides an advanced spectral unmixing on Axioscan 7 devices. It adds the option to define an unmixing directly in the scan profile setup and enables you to create new reference spectra for a dye set. Creating new reference spectra for a dye set may be required if the pre-delivered unmixing library does not cover the dyes (e.g. for not ZEISS-qualified dyes), if a new autofluorescence should be corrected, or if the pre-delivered reference spectra do not allow unmixing results of sufficient quality.

Overview for Creating New Reference Spectra

The Axioscan Advanced Spectral Unmixing module enables you to create new reference spectra. Here is an overview of steps for the creation of reference spectra:

Prepare your reference samples for scanning. For information on how to best prepare the samples, see Sample Preparation for Creating New Reference Spectra.
Acquire reference scans and prepare the images with the usual functionalities of ZEN slidescan. For specific remarks and notes, see Information for Acquisition and Preparation of Reference Scans.
To create your reference spectra, start the Calibrated Unmixing wizard, see Starting the Calibrated Unmixing Wizard.
In the first step, select the autofluorescence spectra, see Selecting Autofluorescence Spectra.
In the second step, select the dye spectra, see Selecting Dye Spectra.
In the third step, check the calculated dye spectra, see Checking the Calculated Dye Spectra.
Once you have created your reference spectra, you can use them for unmixing in your scan profile, just as other reference spectra, see Applying Unmixing in the Scan Profile.

Sample Preparation for Creating New Reference Spectra

When preparing your reference samples, note the following:

Define a dye set of n different dyes that are supposed to be used together in a multiplex scan.
These dyes should be optimized for the fluorescence channels, ensuring minimal possible crosstalk already in the raw data before unmixing.
For each dye of the dye set, one reference sample is required which is a tissue section of a typically used tissue type, stained by only this specific dye (single stained references, no additional nuclear marker).
At least one, unstained, reference sample is required of the same tissue type as the dye reference samples. The unstained sample (no nuclear marker) should be processed as similarly as possible like the single-stained references.
As an example, a set of six dyes (five specific dyes plus a nuclear marker), the following seven reference samples are required:

One sample only stained with dye 1.
One sample only stained with dye 2.
One sample only stained with dye 3.
One sample only stained with dye 4.
One sample only stained with dye 5.
One sample only stained with the nuclear marker.
One sample of the unstained tissue.

It is possible to use the same marker for the single-stained reference slides as for the multiplex samples that will be unmixed later. It is favorable, however, to use the same marker for all dyes to ensure a clear and comparable signal at the same sites of the tissue. It also makes the calibration setup easier.

Information for Acquisition and Preparation of Reference Scans

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.

Starting the Calibrated Unmixing Wizard

You have licensed the Spatial Biology toolkit and activated it in Tools > Toolkit Manager.

Click Tools > Measure Unmixing Spectra.
The Calibrated Unmixing wizard opens.

Selecting Autofluorescence Spectra

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

In the selection field, click .
A file browser opens.
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.
The file is loaded. The image or the spectrum of the selected file is displayed in the Center Screen Area.
In case you have loaded an image, go to the Graphics tab below the image.
The controls for graphical elements are displayed, see Graphics Tab.
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.
You have marked the autofluorescence.
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.
The relevant information is marked in the image.
Go to Select Autofluorescence and select the graphical element that marks the autofluorescence.
Autofluorescence is set.
Go to Select Background and select the graphical element that marks the background, if available. Otherwise select None from the drop-down list.
The background is set.
Click Next.
The next step opens, see Selecting Dye Spectra.

Selecting Dye Spectra

You have selected the autofluorescence spectra and are in the second step of the Calibrated Unmixing wizard, see Selecting Autofluorescence Spectra.
You have prepared reference scans of your sample, see Information for Acquisition and Preparation of Reference Scans.

For Unmixing Database Folder, define a name for the dye set to be used in the unmixing library.
You have defined a name.
Go to the table in the Center Screen Area, select the first entry, and click .
A file browser opens.
Select one of the (cropped) single-stain reference scan images and click Open.
The file name is displayed in the table and the image is displayed below the table for the selected entry.
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.
You have selected the channel.
For Dye, enter a name for the dye for later reference.
Dye name is set.
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.
The channel for autofluorescence is set.
Repeat the previous steps with the single-stain reference scans for all dyes, one per row.
The dye spectra are set.
Click Next.
The next step opens, see Checking the Calculated Dye Spectra.

Checking the Calculated Dye Spectra

You have selected the dye spectra and are in the third step of the Calibrated Unmixing wizard, see Selecting Dye Spectra.

In the chart of the Center Screen Area, check the dye spectra that were calculated based on the input of the previous step.
You have checked the spectra.
To make changes for the spectra, click Back.
The previous step opens and you can adjust your selection to fit your needs, see Selecting Dye Spectra.
If all spectra are satisfactory, click Finish.
All changes are saved and the wizard closes.

You have created new reference spectra that are saved in the unmixing library. You can now use them for unmixing in your scan profile, see Applying Unmixing in the Scan Profile.

Applying Unmixing in the Scan Profile

You have licensed the Spatial Biology toolkit and activated it in Tools > Toolkit Manager.
You are in the Scan step of the Scan Profile Editor.
You have reference spectra available for your unmixing.

In the Online Processing section, activate Unmixing active.
The parameters for unmixing are displayed, see Unmixing Parameters.
For Ref. Spectra, click .
A dialog for the unmixing database opens.
Select the folder containing your reference spectra and click OK.
The reference spectra are loaded.
To change or select a reference file for a channel, go to the table and click for the channel.
A dialog opens.
Select the reference file for the dye of the current channel and click OK.
You have set the reference for the this channel.
Repeat the previous steps for all channels of your experiment.
The reference spectra for your scan are set.
Continue with the remaining setup of your scan profile, see Adjusting Scan Settings and Working in the Scan Profile Editor.