Question: How do I correct the red channel shift observed in the CytAssist image for a Visium HD gene expression assay generated from a CytAssist instrument running firmware version 2.1?
Answer: Update Space Ranger to version 3.1.2 or later and Loupe Browser to version 8.1.2 or later to automatically detect and correct images from CytAssist instruments running software version 2.1.
Visium HD images generated by CytAssist instruments with firmware version 2.1 installed have a shift in the red channel. This only happens to Visium HD CytAssist images and only occurs with firmware version 2.1. The shift can be observed by zooming in on the CyAssist image’s fiducial frame (showen below).
Visium HD CytAssist image with a red channel shift generated by instrument firmware version 2.1
For comparison an image without the shift is provided (shown below). If left uncorrected, this leads to a 4 pixel horizontal misalignment between the gene expression information and the microscope image.
Visium HD CytAssist image without a red channel shift
To verify if the red channel shift affects your Visium HD gene expression analysis, open the “Image Alignment” tab in the web summary file and zoom in on the fiducial frame in the “Tissue Detection and Fiducial Alignment” image. If the red circles around the fiducials are misaligned and shifted 4 pixels to the right, it is likely that the gene expression is misaligned (see the image below for an example of the 4-pixel shift in the red circle).
Fiducial Frame Alignment of a CytAssist image with a red channel shift generated by instrument firmware version 2.1
For images generated with instrument software version 2.1, update Space Ranger to version 3.1.2 or later and Loupe Browser to version 8.1.2 or later. These versions will automatically detect and correct images from CytAssist instruments running software version 2.1. Please contact 10x Technical Support at support@10xgenomics.com for if you require additional assistance with correcting this shift in the red channel.
When a CytAssist image generated from a CytAssist instrument using firmware version 2.1 is corrected and processed by the Space Ranger pipeline, the “Sample Metadata” table in the “Summary” tab of the web summary HTML file will display “corrected” next to the “CytAssist Instrument Software Version” (see the image below where the CytAssist firmware version field is circled in red).
Related knowledge base articles.
- “How do I find the CytAssist instrument firmware version that generated my CytAssist image?”
- “How can I verify the fiducial frame alignment for Visium HD was successful?”
Last Updated: Oct. 2024