# US 11,896,944 B2 · System and method with fiducials responding to multiple excitation frequencies

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- Number: US 11,896,944 B2
- Document ID: US11896944B2
- Status: Granted
- Kind: B2
- Application number: 16/482,592
- Filed: 2018-01-31
- Published: 2020-01-09
- Granted: 2024-02-13
- Earliest claimed: 2017-02-01
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: John S. Vieceli; Alex Nemiroski; Paul Belitz; Robert Langlois; M. Shane Bowen; Danny Yuan Chan; Bala Murali K. Venkatesan; Hui Han; Kevan Samiee; Stephen Tanner
- Web page: https://patentgrep.com/patent/US11896944B2

## CPC

- B01J 19/0046: {Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00)}
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging}
- G06T 7/0012: {Biomedical image inspection}
- B01J 2219/00576: fluorophore
- B01J 2219/00608: DNA chips
- B01J 2219/00648: by the use of solid beads
- B01J 2219/00693: Means for quality control
- B01J 2219/00702: Processes involving means for analysing and characterising the products
- B01J 2219/00722: Nucleotides
- B01L 3/5085: {for multiple samples, e.g. microtitration plates}
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G06T: IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

Fiducial markers are provided on patterned arrays of the type that may be used for molecular analysis, such as sequencing. The fiducials may have configurations that enhance their detection in image or detection data, that facilitate or improve processing, that provide encoding of useful information, and so forth. Examples of the fiducials may include an “always on” type that respond to multiple frequencies of radiation used during processing and detection so as to return signals during successive cycles of imaging.

## Claims

### Claim 1

An array, comprising:
a support having a plurality of areas, each of the plurality of areas having locations that, in operation, receive biological samples differing from one another to respond differently in successive cycles of fluorescent imaging; and
a fiducial feature on the support in each of the plurality of areas, each of the fiducial features including at least one depression and a plurality of autofluorescent beads disposed in the at least one depression, the at least one depression having a shape that separates an inner area where some of the locations are located and an outer area where some other of the locations are located, the shape being selected from the group consisting of a single circle, a single segmented circle, two non-concentric and segmented rings, and a non-closed shape, the autofluorescent beads being formed of a fluorescent material that responds by producing a detectable signal in the successive cycles of fluorescent imaging.

### Claim 2 (depends on 1)

The array of claim 1, further comprising the biological samples in each of the locations, wherein in performing the successive cycles of fluorescent imaging only some of the biological samples appear in successive images to the exclusion of other of the biological samples, but in which the fiducial features appear in all images.

### Claim 3 (depends on 1)

The array of claim 1, wherein the fluorescent material of the fiducial features is responsive to at least two different wavelengths of light used in the successive cycles of fluorescent imaging.

### Claim 4 (depends on 1)

The array of claim 1, wherein each of the plurality of autofluorescent beads disposed in the at least one depression is too large to be received in the locations of the support that receive the biological samples.

### Claim 5 (depends on 1)

The array of claim 1, wherein the locations are disposed in a regular repeating pattern on the support.

### Claim 6 (depends on 1)

The array of claim 1, further comprising the biological samples in each of the locations, wherein the biological samples comprise nucleic acid sequences.

### Claim 7 (depends on 1)

The array of claim 1, wherein the fiducial features are structured to produce image data encoding information.

### Claim 8 (depends on 1)

The array of claim 1, further comprising at least one additional fiducial feature that is formed in or on the support and is optically reflective to, during imaging, return at least a portion of incident radiation for locating, adjusting the location of, or registering the support or the locations.

### Claim 9 (depends on 1)

The array of claim 1, wherein each of the plurality of the autofluorescent beads has a diameter of between about 5 μm and about 10 μm.

### Claim 10 (depends on 1)

The array of claim 1, wherein the fluorescent material is polystyrene, silica, or a combination thereof.

### Claim 11

A method, comprising:
disposing reaction sites at locations on a support including a plurality of areas, each of the plurality of areas having some of the locations, the reaction sites to attach biological samples differing from one another to respond differently in successive cycles of fluorescent imaging; and
disposing a fiducial feature on the support in each of the plurality of areas, the fiducial feature including at least one depression and a plurality of autofluorescent beads disposed in the at least one depression, the at least one depression having a shape that separates an inner area where some of the locations are located and an outer area where some other of the locations are located, the shape being selected from the group consisting of a single circle, a single segmented circle, two non-concentric and segmented rings, and a non-closed shape, the autofluorescent beads being formed of a fluorescent material that responds by producing a detectable signal in the successive cycles of fluorescent imaging.

### Claim 12 (depends on 11)

The method of claim 11, further comprising:
introducing the biological samples to the support to attach the biological samples to the reaction sites; and
imaging the biological samples and the fiducial features in the successive cycles of fluorescent imaging at different wavelengths of light to produce image data that encodes fluorescent signals produced by the biological samples and the fiducial feature.

### Claim 13 (depends on 11)

The method of claim 11, further comprising introducing the biological samples to the support to attach the biological samples to the reaction sites; wherein between each successive cycle of fluorescent imaging, a tag is removed from the biological sample at each location, and an additional biological component is added to each biological sample at each location, the additional biological components having tags that respond to the successive cycle of fluorescent imaging.

### Claim 14 (depends on 1)

The array of claim 1, further comprising an auto-centering fiducial located outside of the plurality of areas on the support where the locations of the support that receive the biological samples are disposed.

### Claim 15 (depends on 7)

The array of claim 7, wherein the information is encoded by alternate signal-producing areas and non-signal producing areas detectable in the image data, and wherein the alternate signal-producing areas and non-signal-producing areas are detectable as unchanged through the successive cycles of fluorescent imaging.

### Claim 16 (depends on 15)

The array of claim 15, wherein the alternate signal-producing areas and non-signal-producing areas comprise dash-like areas, dot-like areas, or combinations thereof.

### Claim 17 (depends on 7)

The array of claim 7, wherein each of the fiducial features comprises a plurality of the autofluorescent objects disposed in the at least one depression, and wherein the information is encoded by a relative orientation or positioning of the autofluorescent objects.

### Claim 18 (depends on 7)

The array of claim 7, wherein each of the fiducial features comprises machine-readable indicia comprising discrete regions arranged to define binary data having an interpretable meaning.

### Claim 19 (depends on 18)

The array of claim 18, wherein the machine-readable indicia comprise a bar-code indicia.

### Claim 20 (depends on 7)

The array of claim 7, wherein the encoded information of one fiducial feature is indicative of an orientation or direction of other fiducial features on the support.

### Claim 21 (depends on 7)

The array of claim 7, wherein the encoded information is indicative of a location of a respective one of the fiducial features.

### Claim 22 (depends on 7)

The array of claim 7, wherein the encoded information is indicative of an identification of the support or a subject donor of the biological samples.