Systems and methods for image registration or alignment

A computer system comprising:

• one or more processors;
• memory;
• and
• one or more programs, wherein the one or more programs are stored in the memory and configured to be executed by the one or more processors, the one or more programs including instructions for overlaying image data for a biological sample onto spatial analyte data for a plurality of analytes of the biological sample by a method comprising:
• obtaining a first image of the biological sample on a first substrate;
• receiving a second image of the biological sample on the first substrate overlayed on a second substrate, wherein the second substrate comprises one or more spatial fiducials and a set of capture spots, and wherein the set of capture spots comprises at least 1000 capture spots,
• wherein at least one of the first substrate and the second substrate is transparent;
• determining a registration for the first image and the second image, wherein the registration registers the first image and the second image to each other, using a first pattern of the biological sample in the first image and a second pattern of the biological sample in the second image;
• and
• using the registration to overlay the first image onto a spatial dataset comprising spatial analyte data for the set of capture spots from the biological sample, wherein a frame of reference of the spatial dataset is known with respect to the second image, based on the one or more spatial fiducials of the second image,
• thereby overlaying image data for the biological sample onto spatial analyte data for the plurality of analytes of the biological sample.

A non-transitory computer readable storage medium storing one or more programs, the one or more programs comprising instructions, which when executed by an electronic device with one or more processors and a memory cause the electronic device to perform a method for overlaying image data for a biological sample onto spatial analyte data for a plurality of analytes of the biological sample, comprising:

• obtaining a first image of the biological sample on a first substrate;
• receiving a second image of the biological sample on the first substrate overlayed on a second substrate, wherein the second substrate comprises one or more spatial fiducials and a set of capture spots, and
• wherein the set of capture spots comprises at least 1000 capture spots, wherein at least one of the first substrate and the second substrate is transparent;
• determining a registration for the first image and the second image, wherein the registration registers the first image and the second image to each other, using a first pattern of the biological sample in the first image and a second pattern of the biological sample in the second image;
• and
• using the registration to overlay the first image onto a spatial dataset comprising spatial analyte data for the set of capture spots from the biological sample, wherein a frame of reference of the spatial dataset is known with respect to the second image, based on the one or more spatial fiducials of the second image,
• thereby overlaying image data for the biological sample onto spatial analyte data for the plurality of analytes of the biological sample.