# US 10,501,788 B2 · System and methods for calibrating binding dyes

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,501,788 B2
- Document ID: US10501788B2
- Status: Granted
- Kind: B2
- Application number: 15/017,034
- Filed: 2016-02-05
- Published: 2016-08-11
- Granted: 2019-12-10
- Earliest claimed: 2015-02-06
- Assignees: Life Technologies Corporation
- Applicants: Life Technologies Corporation
- Inventors: Yong Chu; Jacob Freudenthal; Jeffrey Marks
- Web page: https://patentgrep.com/patent/US10501788B2

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- G01N 21/274: {Calibration, base line adjustment, drift correction}
- 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 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging}
- G01N 2021/6421: {Measuring at two or more wavelengths}
- G01N 2021/6441: {with two or more labels}
- G01N 2201/127: Calibration; base line adjustment; drift compensation
- 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)

## Abstract

Some embodiments describe a computer-implemented method for calibrating a fluorescent dye. The method can comprise imaging a sample holder, loaded into an instrument, at more than one channel. The sample holder can comprise a plurality of reaction sites and more than one dye type, with each dye occupying more than one reaction site. The method can further comprise identifying a peak channel for each dye on the sample holder, normalizing each channel to the peak channel for each dye, and producing a dye matrix that can comprise a set of dye reference values.

## Claims

### Claim 1

A computer-implemented method for calibrating a fluorescent dye, the method comprising:
imaging a sample holder, loaded into an instrument, at more than one channel, the sample holder comprising a plurality of reaction sites and a plurality of dyes comprising more than one dye type, each dye occupying more than one reaction site, wherein the plurality of dyes are distributed in a checkerboard pattern among the reaction sites;
identifying a peak channel for each dye on the sample holder;
normalizing each channel to the peak channel for each dye; and
producing a dye matrix comprising a set of dye reference values.

### Claim 2 (depends on 1)

The computer-implemented method of claim 1, wherein the dyes are distributed in a pattern along the reaction sites such that each dye occupies substantially the same number of reaction sites.

### Claim 3 (depends on 1)

The computer-implemented method of claim 1, wherein the dyes are distributed in a pattern along the reaction sites such that dyes occupying adjacent reaction sites are different.

### Claim 4 (depends on 1)

The computer-implemented method of claim 1, wherein the plurality of dyes are distributed in a repeating alternating pattern along the reaction sites.

### Claim 5 (depends on 1)

The computer-implemented method of claim 1, further comprising:
performing, for a given dye, a first modeling for reaction sites not occupied by the given dye.

### Claim 6 (depends on 5)

The computer-implemented method of claim 5, further comprising:
performing a goodness of fit check on the results of the first modeling.

### Claim 7 (depends on 5)

The computer-implemented method of claim 5, further comprising:
performing, for a given dye, a second modeling for reaction sites not occupied by the given dye.

### Claim 8

A non-transitory computer-readable storage medium encoded with instructions, executable by a processor, for calibrating a fluorescent dye, the instructions comprising instructions for:
imaging a sample holder, loaded into an instrument, at more than one channel, the sample holder comprising a plurality of reaction sites and a plurality of dyes comprising more than one dye type, each dye occupying more than one reaction site, wherein the plurality of dyes are distributed in a checkerboard pattern among the reaction sites;
identifying a peak channel for each dye on the sample holder;
normalizing each channel to the peak channel for each dye; and
producing a dye matrix comprising a set of dye reference values.

### Claim 9 (depends on 8)

The non-transitory computer-readable storage medium of claim 8, wherein the dyes are distributed in a pattern along the reaction sites such that each dye occupies substantially the same number of reaction sites.

### Claim 10 (depends on 8)

The non-transitory computer-readable storage medium of claim 8, wherein the dyes are distributed in a pattern along the reaction sites such that dyes occupying adjacent reaction sites are different.

### Claim 11 (depends on 8)

The non-transitory computer-readable storage medium of claim 8, wherein the plurality of dyes are distributed in a repeating alternating pattern along the reaction sites.

### Claim 12 (depends on 8)

The non-transitory computer-readable storage medium of claim 8, further comprising:
performing, for a given dye, a first modeling for reaction sites not occupied by the given dye.

### Claim 13 (depends on 12)

The non-transitory computer-readable storage medium of claim 12, further comprising:
performing, for a given dye, a second modeling for reaction sites not occupied by the given dye.

### Claim 14

A system for calibrating a fluorescent dye, the system comprising:
a processor; and
a memory encoded with instructions, executable by the processor, the instructions for:
imaging a sample holder, loaded into an instrument, at more than one channel, the sample holder comprising a plurality of reaction sites and a plurality of dyes comprising more than one dye type, each dye occupying more than one reaction site, wherein the plurality of dyes are distributed in a checkerboard pattern among the reaction sites;
identifying a peak channel for each dye on the sample holder;
normalizing each channel to the peak channel for each dye; and
producing a dye matrix comprising a set of dye reference values.

### Claim 15 (depends on 14)

The system of claim 14, wherein the dyes are distributed in a pattern along the reaction sites such that each dye occupies substantially the same number of reaction sites.

### Claim 16 (depends on 14)

The system of claim 14, wherein the dyes are distributed in a pattern along the reaction sites such that dyes occupying adjacent reaction sites are different.

### Claim 17 (depends on 14)

The system of claim 14, wherein the plurality of dyes are distributed in a repeating alternating pattern along the reaction sites.

### Claim 18 (depends on 14)

The system of claim 14, further comprising:
performing, for a given dye, a first modeling for reaction sites not occupied by the given dye.

### Claim 19 (depends on 18)

The system of claim 18, further comprising:
performing a goodness of fit check on the results of the first modeling.

### Claim 20 (depends on 18)

The system of claim 18, further comprising:
performing, for a given dye, a second modeling for reaction sites not occupied by the given dye.