# US 11,029,513 B2 · Dynamic autofocus method and system for assay imager

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- Number: US 11,029,513 B2
- Document ID: US11029513B2
- Status: Granted
- Kind: B2
- Application number: 16/251,859
- Filed: 2019-01-18
- Published: 2019-05-23
- Granted: 2021-06-08
- Earliest claimed: 2008-12-15
- Assignees: Illumina, Inc.
- Applicants: Illumina, Inc.
- Inventors: Darren Robert Segale; John Moon; Hongji Ren
- Web page: https://patentgrep.com/patent/US11029513B2

## CPC

- G02B 26/127: {Adaptive control of the scanning light beam, e.g. using the feedback from one or more detectors (G02B27/0031 takes precedence)}
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- G01B 11/24: for measuring contours or curvatures
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G02B 7/38: measured at different points on the optical axis {, e.g. focussing on two or more planes and comparing image data}
- G02B 21/244: {using image analysis techniques}
- G02B 26/10: Scanning systems
- H04N 23/67: Focus control based on electronic image sensor signals
- H04N 23/672: {based on the phase difference signals}
- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- G01B 9/02063: {by particular alignment of focus position, e.g. dynamic focussing in optical coherence tomography}
- G01N 2015/1452: {Adjustment of focus; Alignment}
- G02B: OPTICAL ELEMENTS, SYSTEMS OR 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
- G01B: MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
- 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)
- H04N: PICTORIAL COMMUNICATION, e.g. TELEVISION

## Abstract

A method for scanning a microarray, including (a) providing a microarray to an optical scanner, wherein the microarray includes a surface having features having different target molecules; (b) scanning the surface in the x or y direction to acquire optical signals from the features at sequential regions of the surface, wherein the focus setting between the optical scanner and the surface is dynamically controlled in real time by repeatedly (i) adding a predetermined focal offset, thereby introducing an error in the focus setting, and (ii) adjusting the focus setting to reduce the error in the focus setting, thereby acquiring the optical signals from different regions at different degrees of focus such that a subset of the regions that are scanned have an introduced focus error; and (c) analyzing the optical signals that are obtained at the different degrees of focus to identify the different target molecules.

## Claims

### Claim 1

An optical imaging system comprising:
a sample holder to receive a sample including a surface having features, wherein the features comprise different target molecules;
an optical assembly to scan the surface in the x or y direction;
a focus control module to dynamically control a focus setting in the z dimension between the optical assembly and the surface in real time, including repeatedly adding a predetermined focal offset in the z dimension, determining a focus score from optical signals that are acquired at the predetermined focal offset, and adjusting the focus setting based on the focus score to reduce an error in the focus setting;
a detector to acquire the optical signals from different regions at different degrees of focus such that a subset of the regions that are scanned have an introduced focus error; and
an image analysis module to analyze the optical signals to identify the different target molecules.

### Claim 2 (depends on 1)

The optical imaging system of claim 1, wherein the focus control module periodically adds the predetermined focal offset.

### Claim 3 (depends on 1)

The optical imaging system of claim 1, wherein the optical signals that are used by the image analysis module are the same optical signals that the focus control module uses to determine the focus score.

### Claim 4 (depends on 1)

The optical imaging system of claim 1, wherein in determining of the focus score the focus control module determines an actual focal plane and a preferred focal plane for individual images of the sample.

### Claim 5 (depends on 1)

The optical imaging system of claim 1, wherein in determining of the focus score the focus control module determines at least two focus scores, and wherein the focus control module adjusts the focus setting based on the at least two focus scores.

### Claim 6 (depends on 1)

The optical imaging system of claim 1, wherein in adjusting of the focus setting the focus control module uses an amplitude and phase of a transition in the focus score.

### Claim 7 (depends on 1)

The optical imaging system of claim 1, wherein the target molecules comprise nucleic acids.

### Claim 8 (depends on 7)

The optical imaging system of claim 7, wherein each of the features comprises an amplified population of a nucleic acid molecule template.

### Claim 9 (depends on 7)

The optical imaging system of claim 7, wherein the nucleic acids are hybridized to probes at the features of the sample.

### Claim 10 (depends on 7)

The optical imaging system of claim 7, wherein the features that comprise the nucleic acids are scanned during cycles of a nucleic acid sequencing reaction.

### Claim 11 (depends on 1)

The optical imaging system of claim 1, wherein the optical assembly performs line scanning.

### Claim 12 (depends on 11)

The optical imaging system of claim 11, wherein the line scanning comprises time delay integration.

### Claim 13 (depends on 1)

The optical imaging system of claim 1, wherein the detector acquires the optical signals as an image of the sample.

### Claim 14 (depends on 13)

The optical imaging system of claim 13, wherein the regions are sequential regions of the sample that are non-overlapping, and wherein the detector acquires a series of adjacent images that are separate and distinct from one another.

### Claim 15

A non-transitory computer-readable medium including instructions that when executed cause operations to be performed, the operations including:
(a) providing a sample to an optical scanner, wherein the sample comprises a surface having features, wherein the features comprise different target molecules;
(b) scanning the surface in the x or y direction to acquire optical signals from the features at sequential regions of the surface, wherein the focus setting in the z dimension between the optical scanner and the surface is dynamically controlled in real time by repeatedly
i. adding a predetermined focal offset in the z dimension,
ii. determining a focus score from the optical signals that are acquired at the predetermined focal offset, and
iii. adjusting the focus setting based on the focus score to reduce an error in the focus setting,
thereby acquiring the optical signals from different regions at different degrees of focus such that a subset of the regions that are scanned have an introduced focus error; and
(c) analyzing the optical signals that are obtained at the different degrees of focus to identify the different target molecules.

### Claim 16 (depends on 15)

The non-transitory computer-readable medium of claim 15, wherein the determining of the focus score comprises determining at least two focus scores and wherein the adjusting of the focus setting is based on the at least two focus scores.

### Claim 17 (depends on 15)

The non-transitory computer-readable medium of claim 15, wherein the adjusting of the focus setting comprises using an amplitude and phase of a transition in the focus score.

### Claim 18 (depends on 15)

The non-transitory computer-readable medium of claim 15, wherein the scanning comprises line scanning.

### Claim 19 (depends on 18)

The non-transitory computer-readable medium of claim 18, wherein the line scanning comprises time delay integration.

### Claim 20 (depends on 15)

The non-transitory computer-readable medium of claim 15, wherein the sequential regions of the sample are non-overlapping and the acquiring of the optical signals at the sequential regions acquires a series of adjacent images that are separate and distinct from one another.