Make Unmixing Matrix allows you to make a unmixing matrix in OMIQ, including Autofluorescence Subtraction, from single stained (single color) controls and apply it to your files.
Looking for how to create and apply a Compensation Matrix? Find that here.
1. Change the default Workflow
If you have made a workflow from the New Default option then you will need to change the default tasks. Delete the Compensation branch by clicking on Actions → Delete → Delete Branch. Click Add Task and select Spectral Unmixing from the task selection. Go back to the Workflow and click Add new child task on the Unmixing task → select Scaling. Your workflow should look like the final image above before you move on to the next step.
2. Add a Scaling Task off the Workflow Root task
Click Add new child task on the Workflow Root task and select Scaling. Adjust your Scaling settings - find out how to scale your data in OMIQ here.
By default, new Workflows go from Workflow Root → Compensation → Scaling... However, you must scale your single color controls prior to performing Make Unmixing Matrix. Therefore, you must make a second branch of your Workflow, as described above.
3. Add Make Unmixing Matrix Task
Click Add new child task on the Scaling task created in Step (2) and select Make Unmixing Matrix from the task selector.
4. Choose your Raw Spectral Channels
Click Chose Channels, select the channels for which you have single color controls for, then click Submit.
5. Configure your Unmixed channels and your Positive and Negative Controls
OMIQ attempts to select these automatically. Click Configure +/- Controls to review the configuration, then click Submit.
6. (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) and Submit.
7. 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 had already created a Lympho AF gate for the Optional AF Subtraction Step (5). We didn't want to limit the single stained controls to just lymphocytes and therefore activated the Root (Unfiltered) cells by Right Clicking → Activate → Click 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 relevant Clean Up Gate.
8. Review Positive and Negative Control Gates
Optionally you can click through your gates and make any necessary adjustments.
9. Calculate the Unmixing Matrix
Click Generate Unmixing Matrix (you may need to scroll down to see this step) → Copy 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 (10) below. Additionally, note that if you exit the Make Unmixing Matrix task, and enter it again, the Unmixing Matrix is not automatically shown. Click the Generate Unmixing Matrix button again to regenerate your Unmixing Matrix.
10. Copy and Apply Unmixing Matrix
Save the Make Unmixing Matrix task → go back to your Workflow → Open the Unmixing task that you created in Step (1) → click ⫶ Actions → Paste. Paste the unmixing matrix that you copied in Step (9) → click Update.
Your unmixing matrix will now be applied to the files indicated in Files in Group. Save the Unmixing task → go back to your Workflow and continue with the downstream tasks.
You can easily view NxN and Spectrum Plots in Figure.