Make Unmixing Matrix allows you to make an unmixing matrix in OMIQ, from single stained (single color) controls and apply it to your files. This process optionally includes Autofluorescence Subtraction.
Looking for how to create and apply a Compensation Matrix? Find that here.
1. Change the default Workflow
A default Workflow in OMIQ can go from Workflow Root → Compensation → Scaling.
Click on Delete on the Compensation task. This should bring the Scaling task up.
The Compensation task may already not be in the default workflow if OMIQ does not detect any spillover matrix in the FCS files. If this is the case, proceed to Step 2 Add Make Unmixing Matrix Task.
2. Add Make Unmixing Matrix Task
Click Add new child task and select Make Unmixing Matrix from the task selector.
3. Choose your Raw Spectral Channels
Click Choose Channels.
Select all the raw channels, then click Submit.
While OMIQ tries to automatically select all the raw channels for you, it is good to double check that all the raw channels were selected and not just the channel where your fluorescent markers peak. The selection of raw channels pertain to all the channels that you wish to use to define the spectrum of your dyes rather than just the peak channels of the dyes. This would not include any non-fluorescent channels (like your scatter channels and time)
4. Configure your Unmixed channels and your Positive and Negative Controls
Click on Configure +/-.
Select the files that are your single stained control files and unstained (as applicable).
Click Submit.
Configure the Output Name (primary), Output Name (secondary), Negative File, and Positive File.
Click Submit.
OMIQ attempts to select these automatically. It is good to review them. In the example above, we are applying a universal negative to all the control files. If desired, you can change the files selected for the Negative File to have a different unstained control for a particular single stained file.
5. (Optional) Autofluorescence Subtraction
On the flow cytometry plot, select the File you want to make any Autofluorescence Gates on and create your gate. Repeat this step if you have multiple Autofluorescence Gates.
Click Configure AF.
Select Your AutoFluorescence Gate(s) and your Reference File (the file from which to collect the AF spectra)
Click Submit.
6. Generate Gates and Set Positions
On the flow cytometry plot, select the File you want to make any clean up gates on and create your gate. In this example we are using single stained cells for our controls and created a Singlets gate (filter). We want to generate our gates on the Singlets gate. To do this, right-click on the node in the Gating Tree and select Activate. Click on Generate Gates.
Note that you can include clean up gates from multiple files and filters. For example, if you wanted to use a mix of control samples that were cells or compensation beads. To do this, perform the step above on the different Files. Activate one of the clean up gates → Click Generate Gates → Right click on the Gate you want to move → Copy Node → Paste Node on the relevant Clean Up Gate.
7. Review Positive and Negative Control Gates
Click through the generated gates and make any necessary adjustments.
8. Calculate the Unmixing Matrix
Click Generate Unmixing Matrix (you may need to scroll down to see this step). This step will generate the Unmixing Matrix.
The Unmixing Matrix you have created is not automatically applied to your data. To apply this Unmixing Matrix to your data, follow the instructions in Step (9) below.
The Unmixing Matrix Condition Number (aka complexity index) is one of the most important quantitative metrics for overall panel quality in spectral cytometry. It measures the uniqueness of each dye in a given panel. The higher the condition number means that there is a higher overall spectral spread among the dyes in the panel. This makes it more challenging to spectrally unmix the dyes in the panel.
Along with the Unmixing Matrix, a Cosine Similarity Matrix and Hotspot Matrix is generated as well.
The Cosine Similarity Matrix shows how unique the spectral signature is of one dye compared to another dye. Values range from 0 (no similarity) to 1 (identical spectra). It is used in spectral panel design to flag fluorochrome pairs with high similarity and predict them as more risky or likely to interfere with each other during unmixing.
The Hotspot Matrix is a useful predictive tool that maps how much each fluorochrome contributes spread into others. High-value cells are called 'hotspots' and highlight problematic dye pairings likely to cause spectral overlap, unmixing-dependent spread (UDS), and reduced resolution, especially for low-expression markers.
Note that if you exit the Make Unmixing Matrix task, and enter it again, the Unmixing Matrix, Cosine Similarity Matrix, and Hotspot Matrix are not automatically shown. Click the Generate Unmixing Matrix button again to regenerate these matrices again.
9. Copy and Apply Unmixing Matrix
Click on Copy Matrix.
Remember to save the task by clicking on Save Task.
Click on Back to Workflow to exit the Make Unmixing Task.
On the Workflow Root, click on Add new child task. Select Spectral Unmixing from the task selector.
Click on Create New Unmixing Group.
Choose the File(s) that you would want to unmix together using the Unmixing Matrix generated in Step (8).
Click Submit.
Click on Actions.
Select Paste Matrix.
Paste the Unmixing Matrix and click Update.
The Unmixing Matrix is now applied to the files within the Unmixing Group. Click on Save Task and return to the workflow to continue with your analysis.
Remember to add a Scaling task as a child of the Unmixing Task and check how the scaling looks on the unmixed features. To learn more about data scaling, please read our Understanding Data Scaling article.
You can easily view NxN and Spectrum Plots in Figure.