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EP-4740054-A1 - SYSTEMS AND METHODS FOR TISSUE BOUNDS DETECTION

EP4740054A1EP 4740054 A1EP4740054 A1EP 4740054A1EP-4740054-A1

Abstract

Provided herein are methods for minimizing Z-image acquisition comprising receiving a first set of three-dimensional (3D) positional information of a plurality of biological molecules within a sample, wherein the first set of 3D positional information is within a first imaging volume and based on a probing cycle of the sample in an imaging instrument, wherein the probing cycle comprises generating optical signals corresponding to at least some of the plurality of biological molecules; determining, based on the first set of 3D positional information, a second imaging volume that is less than the first imaging volume; and directing the imaging instrument to image the second imaging volume in at least one subsequent probing cycle of the sample.

Inventors

  • SKRYNNYK, OLEKSIY
  • SMIECHOWSKI, ADAM HENRYK
  • MONKOWSKI, Adam Joseph
  • HEIBERG, ANDREW
  • HOFFMAN, DAVID

Assignees

  • 10X Genomics, Inc.

Dates

Publication Date
20260513
Application Date
20240703

Claims (20)

  1. 1. A method comprising: receiving a first set of three-dimensional (3D) positional information of a plurality of biological molecules within a sample, wherein the first set of 3D positional information is within a first imaging volume and based on a probing cycle of the sample in an imaging instrument, wherein the probing cycle comprises generating optical signals corresponding to at least some of the plurality of biological molecules; determining, based on the first set of 3D positional information, a second imaging volume that is less than the first imaging volume; and directing the imaging instrument to image the second imaging volume in at least one subsequent probing cycle of the sample.
  2. 2. The method of claim 1, further comprising: receiving a plurality of images from the probing cycle, wherein the plurality of images comprises the plurality of optical signals; determining, based on the plurality of optical signals, the first set of 3D positional information.
  3. 3. The method of claim 2, wherein the plurality of images comprises a plurality of z- stacks of the sample.
  4. 4. The method of claim 3, wherein a distance between focal planes in the plurality of z- stacks is about 0.25 pm to about 1 pm.
  5. 5. The method of claim 4, wherein the distance is about 0.75 pm.
  6. 6. The method of any one of claims 2 to 5, wherein determining the first set of 3D positional information comprises blob detection of the plurality of optical signals.
  7. 7. The method of any one of claims 2 to 6, wherein determining the first set of 3D positional information comprises image registration and/or alignment of the plurality of focal planes in each z-stack of the plurality of z-stacks.
  8. 8. The method of any one of claims 1 to 7, further comprising receiving a second set of 3D positional information of at least a portion of the plurality of biological molecules, wherein the second set of 3D positional information is obtained from a probing cycle of the at least one subsequent probing cycle.
  9. 9. The method of any one of claims 1 to 8, wherein the biological sample defines a sample volume, and wherein the first imaging volume is greater than the sample volume.
  10. 10. The method of claim 9, wherein the second imaging volume is equal to the sample volume.
  11. 11. The method of claim 9, wherein the second imaging volume is less than the sample volume.
  12. 12. The method of claim 9, wherein the second imaging volume is greater than the sample volume.
  13. 13. The method of any one of claims 1 to 12, further comprising: determining, based on the second set of 3D positional information, a third imaging volume that is greater than or less than the second imaging volume.
  14. 14. The method of claim 13, directing the imaging instrument to image the third imaging volume in at least one subsequent probing cycle of the sample.
  15. 15. The method of any one of claims 1 to 14, wherein determining the second imaging volume comprises truncating the first set of 3D positional information.
  16. 16. The method of claim 15, wherein about 0.01% to about 1% of the first set of 3D positional information is truncated at each end.
  17. 17. The method of claim 16, wherein about 0.2% of the first set of 3D positional information is truncated at each end.
  18. 18. The method of any one of claims 1 to 17, wherein the first imaging volume has a height of about 25 pm to about 50 pm.
  19. 19. The method of claim 18, wherein the first imaging volume has a height of about 30 pm.
  20. 20. The method of any one of claims 1 to 19, wherein the biological sample has a thickness of about 5 pm to about 20 pm.

Description

SYSTEMS AND METHODS FOR TISSUE BOUNDS DETECTION Cross-Reference to Related Applications [0001] This application claims the benefit of U.S. Provisional Application No. 63/525,540, filed July 7, 2023, which is hereby incorporated by reference in its entirety. Field of the Invention [0002] The present disclosure is directed to imaging techniques for samples, e.g., biological samples. More specifically, the present disclosure describes methods for identifying the Z- bounds of a sample based on a signal of interest detected within the sample, and minimizing an imageable volume of said sample to reduce imaging time and/or maximize throughput while retaining data quality. Background [0003] In situ detection and analysis methods are emerging from the rapidly developing field of spatial transcriptomics. The key objectives in spatial transcriptomics are to detect, quantify, and map gene activity to specific regions in a tissue sample at cellular or sub- cellular resolution. These techniques allow one to study the subcellular distribution of gene activity (as evidenced, e.g., by expressed gene transcripts), and have the potential to provide crucial insights in the fields of developmental biology, oncology, immunology, histology, etc. [0004] In situ decoding is a process comprising a plurality of decoding cycles in each of which a different set of barcode probes (e.g., fluorescently-labeled oligonucleotides) is contacted with target analytes (e.g., mRNA sequences) or with target barcodes (e.g., nucleic acid barcodes) associated with the target analytes present in a sample (e.g., a tissue sample) under conditions that promote hybridization. One or more images (e.g., fluorescence images) are acquired in each decoding cycle, and the images are processed to detect the presence and locations of one or more barcode probes in each cycle. The presence and locations of one or more target analyte sequences or associated barcode sequences are then inferred from corresponding code words that are determined based on the set of, e.g., fluorescence signals detected in each decoding cycle of the decoding process. [0005] Accordingly, there exists a need for fast and accurate Z-dimension image acquisition and analysis methods for use in automated and high-throughput imaging systems. Summary [0006] In some aspects, provided herein are methods comprising receiving a first set of three- dimensional (3D) positional information of a plurality of biological molecules within a sample, wherein the first set of 3D positional information is within a first imaging volume and based on a probing cycle of the sample in an imaging instrument, wherein the probing cycle comprises generating optical signals corresponding to at least some of the plurality of biological molecules; determining, based on the first set of 3D positional information, a second imaging volume that is less than the first imaging volume; and directing the imaging instrument to image the second imaging volume in at least one subsequent probing cycle of the sample. [0007] In some aspects of the invention as provided herein, is a computer program product comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform a method comprising receiving a first set of 3D positional information of a plurality of biological molecules within a sample, wherein the first set of 3D positional information is within a first imaging volume and based on a probing cycle of the sample in an imaging instrument, wherein the probing cycle comprises generating optical signals corresponding to at least some of the plurality of biological molecules; determining, based on the first set of 3D positional information, a second imaging volume that is less than the first imaging volume; and directing the imaging instrument to image the second imaging volume in at least one subsequent probing cycle of the sample. [0008] Further aspects of the invention include a system comprising a database; and a computing node comprising a computer readable storage medium having program instructions embodied therewith, the program instructions executable by a processor to cause the processor to perform a method comprising receiving, from the database, a first set of 3D positional information of a plurality of biological molecules within a sample, wherein the first set of 3D positional information is within a first imaging volume and based on a probing cycle of the sample in an imaging instrument, wherein the probing cycle comprises generating optical signals corresponding to at least some of the plurality of biological molecules; determining, based on the first set of 3D positional information, a second imaging volume that is less than the first imaging volume; and directing the imaging instrument to image the second imaging volume in at least one subsequent probing cycle of the sample. [0009] In certain aspects o