Granted Patent B2

US 12,731,273 · App. 18/692,758 · Granted Sep 8, 2026

Inventors: Olga Vorobyova (Pleasanton, CA); Brynn Claypoole (Pleasanton, CA); Dongyao Li (Pleasanton, CA); Neil Ira Weisenfeld (Pleasanton, CA); Didem Pelin Sarikaya (Pleasanton, CA); Peigeng Li (Pleasanton, CA); Guy Joseph (Pleasanton, CA); Eric Siegel (Pleasanton, CA); Naga Sudha Kodavatikanti (Cupertino, CA)

Assignee: 10x Genomics, Inc.

G06T7/337G06T7/13G06T2207/10024G06T2207/10056G06T2207/10064G06T2207/20036G06T2207/20101G06T2207/30024

Abstract

Claims (60)

1 . A method for overlaying image data for a biological sample onto spatial analyte data for a plurality of analytes of the biological sample, the method comprising:

using a computer system comprising one or more processing cores and a memory:

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.

2 . The method of claim 1 , wherein the first image of the biological sample is a histological image of the biological sample.

3 . The method of claim 1 , wherein the determining the registration for the first image and the second image comprises:

identifying one or more landmarks, each respective landmark comprising a respective pair of reference positions including a first respective reference position for the first pattern of the biological sample in the first image and a corresponding second respective reference position for the second pattern of the biological sample in the second image, thereby identifying a first set of landmark coordinates for the first image and a second set of landmark coordinates for the second image;

using the first set of landmark coordinates for the first image and the second set of landmark coordinates for the second image to obtain a transformation between the first set of landmark coordinates and the second set of landmark coordinates with an alignment algorithm, thereby obtaining a first alignment of the first image with the second image.

4 . The method of claim 3 , wherein the identifying one or more landmarks is performed, on a display, via manual user selection of each respective pair of reference positions in the first image and the second image.

5 . The method of claim 3 , further comprising performing, on a display, via user interaction, a manual alignment of the first image and the second image.

6 . The method of claim 5 , wherein the manual alignment is selected from the group consisting of: a translation, a scaling, and a rotation.

7 . The method of claim 3 , further comprising refining the transformation using a refinement algorithm, thereby obtaining a second alignment of the first image with the second image.

8 . The method of claim 3 , wherein a respective similarity metric for a respective alignment of the first image with the second image is determined by evaluating an intensity of a first plurality of pixels in the first image compared to corresponding pixels in a second plurality of pixels in the second image that are overlayed onto each other in the respective alignment.

9 . The method of claim 3 , wherein the one or more landmarks comprises 3 landmarks.

10 . The method of claim 3 , wherein the alignment algorithm transforms the first set of landmark coordinates for the first image relative to the second set of landmark coordinates for the second image.

11 . The method of claim 3 , wherein the alignment algorithm transforms the second set of landmark coordinates for the second image relative to the first set of landmark coordinates for the first image.

12 . The method of claim 3 , further comprising displaying, on a display, an image overlay for the first image aligned with the second image, wherein the first image is displayed in color and the second image is displayed in grayscale.

13 . The method of claim 1 , wherein the determining the registration for the first and second image comprises:

obtaining a respective first area of the first pattern and a respective first centroid for the first area from an analysis of the first pattern;

obtaining a respective second area of the second pattern and a respective second centroid for the second area from an analysis of the second pattern;

scaling at least one of the first image and the second image based on a relative proportion of the first and second area;

centering the first image with the second image based on an alignment between the first and second centroid;

sampling, for each respective rotation angle in a plurality of rotation angles, a corresponding transformation between the first and second image, by a procedure that comprises:

rotating one of the first and second image relative to the other of the first and second image by the respective rotation angle to obtain a respective candidate rotational alignment of the first and second image;

refining the respective candidate rotational alignment to obtain a respective refined candidate rotational alignment; and

scoring the respective refined candidate rotational alignment of the first and second image by determining a similarity metric across a respective intensity of a first plurality of pixels in the first image compared to corresponding pixels in a second plurality of pixels in the second image that are overlayed onto each other by the respective candidate rotational alignment of the first and second image,

thereby obtaining a plurality of refined candidate rotational alignments and a corresponding plurality of similarity metrics; and

selecting the rotational alignment from among the plurality of refined candidate rotational alignments that yields the highest similarity metric in the corresponding plurality of similarity metrics as the registration for the first and second image.

14 . The method of claim 13 , wherein the plurality of rotation angles comprises 4 or more different rotation angles, wherein each rotation angle in the plurality of rotation angles is between 0 and 2π radians.

15 . The method of claim 13 , wherein the procedure further comprises:

rotating one of a mirror image of the first image and the second image relative to the other of the mirror image of the first image and the second image by the respective rotation angle to obtain a respective candidate rotational alignment of the mirror image of the first image and the second image; and

scoring the respective candidate rotational alignment of the mirror image of the first image and the second image by determining a similarity metric for the respective candidate rotational alignment of the mirror image of the first image and the second image.

16 . The method of claim 1 , wherein:

the first image is obtained without spatial fiducials,

the determining a registration for the first image and the second image comprises overlaying the one or more spatial fiducials of the second image onto the first image, and

the using the registration to overlay the first image onto the spatial dataset comprises associating the one or more spatial fiducials of the first image to the frame of reference of the spatial dataset, thereby allowing the first image to be in the same frame of reference as the spatial dataset.

17 . The method of claim 1 , wherein the first image and the second image have different image resolutions.

18 . The method of claim 1 , wherein the biological sample is prepared for imaging on the first substrate using a stain selected from the group consisting of: live/dead stain, trypan blue, periodic acid-Schiff reaction stain, Masson's trichrome, Alcian blue, van Gieson, reticulin, Azan, Giemsa, Toluidine blue, isamin blue, sudan black and osmium, acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, safranin, or a combination thereof.

19 . The method of claim 1 , wherein the first image is obtained when the sample is not in contact with the second substrate and wherein the second image is obtained when the biological sample is in contact with the second substrate.

20 . 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.

21 . 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.

Assignments (1)

Continuity (3)

References Cited (84)