Question: My fiducial frame is not visible in the IF image and I want to keep the image adjustments as is. How do I supply an auxiliary image for manual tissue alignment in Loupe?
Answer: Make a copy of the current IF image and using microscopy software, e.g. Fiji, adjust contrast and brightness to make the frame visible and save. Use this twin image as the auxiliary image in Loupe manual tissue alignment.
For immunofluorescence (IF), spaceranger does not perform automatic tissue detection and so you will have to perform manual tissue alignment using Loupe Browser. Instructions are at https://support.10xgenomics.com/spatial-gene-expression/software/visualization/latest/alignment. In particular, we describe the auxiliary image as follows.
In the panel where you supply Loupe your main image is an option to supply the auxiliary image. The auxiliary image becomes necessary if Loupe's brightness and contrast adjustment ranges are insufficient to visualize the fiducial frame. Note, image adjustments you make within the Manual Alignment Wizard do not impact downstream analysis. You will provide spaceranger the same image file, yes, but Loupe does not change nor save any pixel adjustments back to the original image file. The manual alignment process produces a JSON format text file containing frame and spot coordinates. So, first try increasing brightness and decreasing contrast within Loupe. If your main image then reveals the fiducial frame, then great. If not, read on.
Some researchers find adjusting their monitor brightness and contrast settings helps to reveal faint fiducial frames. For example, I can see some image fiducial spots on my MacBook laptop screen, where brightness is maximal, but I do not see them on my external monitor, where I have set brightness and contrast low so as not to strain my eyes.
If however the image adjustment ranges within Loupe are insufficient for you to visualize the fiducial frame, then you will create an auxiliary image. Duplicate the image file and open it in Fiji or your favorite image processing software. We describe Fiji at https://kb.10xgenomics.com/hc/en-us/articles/4412795731085. Then adjust the image brightness and contrast and save the changed pixel values. Load this image into Loupe as the auxiliary image. On the next page, you may have to select the auxiliary image from the dropdown menu that appears when you click 'Page'.
last modified: 1/27/2022
tags: Visium, Visium for FFPE