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US 2022/0065787 A1Published application
Methods of mfish using alignment beads
AssigneeApplied Materials, Inc.
InventorsChloe Kim, Lu Yan
Life of this patent
Estimating the term…
Abstract
This disclosure relates to methods for image registration in fluorescence assays for the detection and quantitation of analytes in a sample using functionalized alignment beads as fiducial markers to improve image registration.
Claims25
A method for determining the relative location of a biological analyte within a biological sample, the method comprising:
- receiving a biological sample on a support, the sample having a first nucleotide sequence and a second nucleotide sequence or having a first and second pluralities of targeting probes that bind to targeted nucleotide sequences on the sample with the first plurality of targeting probes having the first nucleotide sequence and the second plurality of targeting probes having the second nucleotide sequence;
- receiving a plurality of beads on the support, each bead having a plurality of binding sites, a first subset of the plurality of binding sites including the first nucleotide sequence and a second subset of the plurality of binding sites the second nucleotide sequence;
- exposing the sample and plurality of beads to a first plurality of first probes, each first probe having a fluorophore and a complementary first nucleotide sequence such that the complementary first nucleotide sequence binds to the first nucleotide sequences on the beads and first nucleotide sequences in the sample or in the first plurality of targeting probes;
- obtaining a first image of the sample and the plurality of beads;
- deactivating the first plurality of first probes;
- subjecting the sample and plurality of beads with a second plurality of second probes, each second probe having a fluorophore and a complementary second nucleotide sequence such that the complementary second nucleotide sequence binds to the second nucleotide sequence on the beads and the second nucleotide sequence in the sample or in the second plurality of targeting probes;
- obtaining a second image of the sample and the plurality of beads;
- detecting locations of the plurality of beads in the first image and the second image;
- and
- performing a registration of the first image and the second image based on the detected locations.
7 more elements
A method for performing an in situ fluorescence hybridization assay on a sample, the method comprising:
- a) contacting the sample with one or more targeting-probes, wherein each targeting-probe binds to an analyte in the sample, if present;
- b) contacting the sample with a plurality of fiducial markers, wherein each fiducial marker comprises a plurality of binding sites;
- c) contacting the sample with one or more readout-probes, wherein each readout-probe independently comprises a fluorescent moiety, and wherein each readout probe binds with the one or more targeting probes, if present, and the plurality of binding sites, thereby exhibiting one or more fluorescent signals;
- d) imaging the one or more fluorescence signals produced by each readout-probe;
- e) registering the image in step d);
- f) Repeating steps (a)-(e) from 0 to 10 times;
- g) Photobleaching the sample;
- and
- h) Independently repeating steps (a)-(g) from 0 to 10 times.
6 more elements
Dependencies are inferred from the claim text.