Question: Do misprinted fiducial frame spots affect the performance of automated image alignment?
Answer: The automated fiducial detection and alignment procedure maps molecular barcodes to coordinates in the customer-supplied image by identifying a pattern of fiducial marks printed in a “frame” around the tissue capture area; referred to as the fiducial frame as presented below.
Due to the nature of the fiducial frame, positions for each individual frame spot can deviate from a perfect pattern. This does not, however, affect the relationship between the frame in its entirety and the capture area spots in the interior. The highly accurate interior spot printing and the correct alignment of the spot coordinates to the customer-supplied image during data processing are still maintained even if some frame spots appear misplaced.
Figure 1 shows a section of an image produced during slide manufacturing and demonstrates how, even if a portion of the frame is misprinted, this has little if any effect on our ability to localize capture area spots in the interior.
Figure 1: A portion of a poorly printed fiducial frame imaged using a technique that illuminates both the fiducial frame and the interior capture spots. The red circles demonstrate the mapping that results from the alignment process, with the misprinted circles largely ignored. The capture area spots interior to the frame are not used for alignment, but instead, they demonstrate how the alignment of a partially misprinted fiducial frame leads to the interior spots still being well localized, with only minor positional errors around an idealized center for each spot.
The fiducial alignment process utilizes an algorithm that attempts to derive the best fit rigid alignment between the idealized fiducial frame pattern and the pattern observed in the customer-supplied image. This algorithm does not make explicit choices about which spot is which, but iterates between estimating probabilities and estimating a coordinate transform. Misprints have little effect on the result because they make up a small fraction of the fiducial frame and they are inconsistent with patterns provided by the other fiducial spots, all of which must come together to form an overall square shape. The evidence for the misprints in Figure 1 mostly supports moving the frame inward from left to right, but any possible change that could accommodate this would cause misalignment of so many other spots, that the shift related to the minor misprint is discounted.
Internally, our experience suggests that with proper imaging and accurate stitching, the current automatic fiducial alignment fails only in cases where much of the fiducial frame is blocked by tissue. If you’re experiencing alignment issues where the cause is less certain, we kindly request you share examples with us at firstname.lastname@example.org.
Product: Visium, Visium for FFPE