US 2011/0301062 A1 · RELIABLE FLUORESCENCE CORRECTION METHOD FOR TWO-COLOR MEASUREMENT FLUORESCENCE SYSTEM

Abstract

Methods and devices use in two-color measurement systems. The methods and devices include methods of making corrections, methods of calculating correction factors, fluorescence scanners, and microarray chips. The said methods and devices enable a user to correct fluorescence intensities for errors caused by the occurrence of FRET and /or cross-talk when two fluorophores are used in two-color fluorescence arrays.

Claims18

Dependent claims

A microarray chip for use in a two-color fluorescence measurement system, comprising three kinds of standard spots that are useful for determining correction factors for fluorescence measurements with the system, wherein the three standard spots comprise:

standard spot 1 containing only the donor fluorophore and none of the acceptor fluorophore used in the two-color fluorescence measurement system,
standard spot 2 containing only the acceptor fluorophore and none of the donor fluorophore used in the two-color fluorescence measurement system,
where the acceptor fluorophore in standard spot 2 is present in the same molar amount as the donor fluorophore in standard spot 1, and
standard spot 3 containing the donor and acceptor fluorophores together, wherein the molar quantity of the donor fluorophore in standard spot 3 equals the molar quantity of the acceptor fluorophore in standard spot 3.

1 more element

A microarray fluorescence scanner that resolves fluorescence as a function of spatial coordinates in two dimensions, comprising:

a first fluorescence observation channel that provides excitation at a wavelength adapted for a first fluorophore, and a detector configured to detect the emission wavelength characteristic of the first fluorophore;
a second fluorescence observation channel that provides excitation at a wavelength adapted for a second fluorophore, and a detector configured to detect the emission wavelength characteristic of the second fluorophore;
and
a third fluorescence observation channel that provides excitation at a wavelength adapted for the first fluorophore, and a detector configured to detect the emission wavelength characteristic of the second fluorophore.

1 more element

A method to correct measured fluorescence emissions for the occurrence of cross-talk in a two-color microarray measurement using a donor fluorophore and an acceptor fluorophore, comprising:

(1) measuring the fluorescence intensity for standard spot 1 containing only the donor fluorophore, and measuring the fluorescence intensity for standard spot 2 containing only the acceptor fluorophore;
(2) Determining cross-talk factors φ, β, φ and φ, where:
φ is the ratio of the signal obtained from acceptor channel to the signal obtained from FRET channel in standard spot 1 by the fluorescence scanner;
β is the ratio of the signal obtained from donor channel to the signal obtained from FRET channel in standard spot 1 by the fluorescence scanner;
φ is the ratio of the signal obtained from donor channel to the signal obtained from FRET channel in standard spot 2 by the fluorescence scanner;
and
α is the ratio of the signal obtained from acceptor channel to the signal obtained from FRET channel in standard spot 2 by the fluorescence scanner;
and
(3) Measuring fluorescence of each sample spot on the microarray chip with donor channel to determine MDD, with acceptor channel to determine MAA, and with FRET channel to determine MDA;
and measuring fluorescence of the standard spot 3 on the microarray chip with donor channel to determine M′DD, with acceptor channel to determine M′AA, and with FRET channel to determine M′DA;
and
(4) Calculating the actual direct fluorescence of each sample spot on the microarray chip corrected for cross-talk by these equations:
I
SensitizedAcceptor
=
M
DA
·
α
·
β
-
φ
·
ϕ
(
α
-
φ
)
·
(
β
-
ϕ
)
-
M
DD
·
1
(
β
-
ϕ
)

M
AA
·
1
(
α
-
φ
)
(
37
)

I
Acceptor
=
M
AA
·
α
(
α
-
φ
)
-
M
DA
·
α
·
φ
(
α
-
φ
)



and
(
38
)

I
Donor
=
M
DD
·
β
(
β
-
ϕ
)
-
M
DA
·
β
·
ϕ
(
β
-
ϕ
)
(
39
)
and calculating the actual direct fluorescence of standard spot 3 on the microarray chip corrected for cross-talk by these equations:
I
SensitizedAcceptor
′
=
M
DA
′
·
α
·
β
-
φ
·
ϕ
(
α
-
φ
)
·
(
β
-
ϕ
)
-
M
DD
′
·
1
(
β
-
ϕ
)
-
M
AA
′
·
1
(
α
-
φ
)
(
40
)

I
Acceptor
′
=
M
AA
′
·
α
(
α
-
φ
)
-
M
DA
′
·
α
·
φ
(
α
-
φ
)



and
(
41
)

I
Donor
′
=
M
DD
′
·
β
(
β
-
ϕ
)
-
M
DA
′
·
β
·
ϕ
(
β
-
ϕ
)
(
42
)
where ISensitizedAcceptor is the actual sensitized acceptor fluorescence of each sample spot on the microarray chip due to FRET;
IDonor is the actual direct donor fluorescence of each sample spot on the microarray chip;
IAcceptor is the actual direct acceptor fluorescence of each sample spot on the microarray chip;
I′SensitizedAcceptor is the actual sensitized acceptor fluorescence of standard spot 3 due to FRET on the microarray chip;
I′Donor is the actual direct donor fluorescence of standard spot 3 on the microarray chip;
and I′Acceptor is the actual direct acceptor fluorescence of standard spot 3 on the microarray chip.

240 more elements

A method for determining a detection-correction factor G, which is a system parameter that is characteristic of a fluorescence scanner for measuring fluorescence in two-color microarray measurements, comprising using the actual fluorescence values I′SensitizedAcceptor, I′Acceptor and I′Donor for standard spot 3, wherein the detection-correction factor G is calculated according to the following equation:

G
=
I
SensitizedAcceptor
′
I
Acceptor
′
γ
-
I
Donor
′
(
53
)
where I′SensitizedAcceptor is the actual sensitized acceptor fluorescence of standard spot 3;
I′Donor is the actual direct donor fluorescence of standard spot 3;
I′Acceptor is the actual direct acceptor fluorescence of standard spot 3;
and the transfer factor γ is determined from the relationship:
γ
=
I
acc
I
don
(
54
)
where Iacc is the acceptor fluorescence of standard spot 2, and Idon is the donor fluorescence of standard spot 1;
wherein standard spots 1, 2 and 3 are standard spots of known composition on a chip, which may be the same chip as the microarray chip or a different chip, and where
standard spot 1 contains only the donor fluorophore and none of the acceptor fluorophore used in the two-color fluorescence measurement system,
standard spot 2 contains only the acceptor fluorophore and none of the donor fluorophore used in the two-color fluorescence measurement system,
where the acceptor fluorophore in standard spot 2 is present in the same molar amount as the donor fluorophore in standard sample 1, and
standard spot 3 contains the donor and acceptor fluorophores together, wherein the molar quantity of the donor fluorophore in standard spot 3 equals the molar quantity of the acceptor fluorophore in standard spot 3.

32 more elements

Dependencies are inferred from the claim text.

Facts

Application

12/997,223Filed 11 Jun 2008

Published

8 Dec 2011

Term ends

Not estimatedThe source records don’t support an estimate.

Classification5 codes

G01N 21/278
Calibration, base line adjustment, drift correction: constitution of standards
G01N 21/6452
Specially adapted constructive features of fluorimeters: individual samples arranged in a regular 2D-array, e.g. multiwell plates
G01N 2021/6421
Spectrofluorimetric devices: measuring at two or more wavelengths
G01N 2021/6441
With indicators, stains, dyes, tags, labels, marks: with two or more labels

Abstract

Claims18

Dependent claims

standard spot 1 containing only the donor fluorophore and none of the acceptor fluorophore used in the two-color fluorescence measurement system,
standard spot 2 containing only the acceptor fluorophore and none of the donor fluorophore used in the two-color fluorescence measurement system,
where the acceptor fluorophore in standard spot 2 is present in the same molar amount as the donor fluorophore in standard spot 1, and
standard spot 3 containing the donor and acceptor fluorophores together, wherein the molar quantity of the donor fluorophore in standard spot 3 equals the molar quantity of the acceptor fluorophore in standard spot 3.

1 more element

A microarray fluorescence scanner that resolves fluorescence as a function of spatial coordinates in two dimensions, comprising:

a first fluorescence observation channel that provides excitation at a wavelength adapted for a first fluorophore, and a detector configured to detect the emission wavelength characteristic of the first fluorophore;
a second fluorescence observation channel that provides excitation at a wavelength adapted for a second fluorophore, and a detector configured to detect the emission wavelength characteristic of the second fluorophore;
and
a third fluorescence observation channel that provides excitation at a wavelength adapted for the first fluorophore, and a detector configured to detect the emission wavelength characteristic of the second fluorophore.

1 more element

A method to correct measured fluorescence emissions for the occurrence of cross-talk in a two-color microarray measurement using a donor fluorophore and an acceptor fluorophore, comprising:

(1) measuring the fluorescence intensity for standard spot 1 containing only the donor fluorophore, and measuring the fluorescence intensity for standard spot 2 containing only the acceptor fluorophore;
(2) Determining cross-talk factors φ, β, φ and φ, where:
φ is the ratio of the signal obtained from acceptor channel to the signal obtained from FRET channel in standard spot 1 by the fluorescence scanner;
β is the ratio of the signal obtained from donor channel to the signal obtained from FRET channel in standard spot 1 by the fluorescence scanner;
φ is the ratio of the signal obtained from donor channel to the signal obtained from FRET channel in standard spot 2 by the fluorescence scanner;
and
α is the ratio of the signal obtained from acceptor channel to the signal obtained from FRET channel in standard spot 2 by the fluorescence scanner;
and
(3) Measuring fluorescence of each sample spot on the microarray chip with donor channel to determine MDD, with acceptor channel to determine MAA, and with FRET channel to determine MDA;
and measuring fluorescence of the standard spot 3 on the microarray chip with donor channel to determine M′DD, with acceptor channel to determine M′AA, and with FRET channel to determine M′DA;
and
(4) Calculating the actual direct fluorescence of each sample spot on the microarray chip corrected for cross-talk by these equations:
I
SensitizedAcceptor
=
M
DA
·
α
·
β
-
φ
·
ϕ
(
α
-
φ
)
·
(
β
-
ϕ
)
-
M
DD
·
1
(
β
-
ϕ
)

M
AA
·
1
(
α
-
φ
)
(
37
)

I
Acceptor
=
M
AA
·
α
(
α
-
φ
)
-
M
DA
·
α
·
φ
(
α
-
φ
)



and
(
38
)

I
Donor
=
M
DD
·
β
(
β
-
ϕ
)
-
M
DA
·
β
·
ϕ
(
β
-
ϕ
)
(
39
)
and calculating the actual direct fluorescence of standard spot 3 on the microarray chip corrected for cross-talk by these equations:
I
SensitizedAcceptor
′
=
M
DA
′
·
α
·
β
-
φ
·
ϕ
(
α
-
φ
)
·
(
β
-
ϕ
)
-
M
DD
′
·
1
(
β
-
ϕ
)
-
M
AA
′
·
1
(
α
-
φ
)
(
40
)

I
Acceptor
′
=
M
AA
′
·
α
(
α
-
φ
)
-
M
DA
′
·
α
·
φ
(
α
-
φ
)



and
(
41
)

I
Donor
′
=
M
DD
′
·
β
(
β
-
ϕ
)
-
M
DA
′
·
β
·
ϕ
(
β
-
ϕ
)
(
42
)
where ISensitizedAcceptor is the actual sensitized acceptor fluorescence of each sample spot on the microarray chip due to FRET;
IDonor is the actual direct donor fluorescence of each sample spot on the microarray chip;
IAcceptor is the actual direct acceptor fluorescence of each sample spot on the microarray chip;
I′SensitizedAcceptor is the actual sensitized acceptor fluorescence of standard spot 3 due to FRET on the microarray chip;
I′Donor is the actual direct donor fluorescence of standard spot 3 on the microarray chip;
and I′Acceptor is the actual direct acceptor fluorescence of standard spot 3 on the microarray chip.

240 more elements

G
=
I
SensitizedAcceptor
′
I
Acceptor
′
γ
-
I
Donor
′
(
53
)
where I′SensitizedAcceptor is the actual sensitized acceptor fluorescence of standard spot 3;
I′Donor is the actual direct donor fluorescence of standard spot 3;
I′Acceptor is the actual direct acceptor fluorescence of standard spot 3;
and the transfer factor γ is determined from the relationship:
γ
=
I
acc
I
don
(
54
)
where Iacc is the acceptor fluorescence of standard spot 2, and Idon is the donor fluorescence of standard spot 1;
wherein standard spots 1, 2 and 3 are standard spots of known composition on a chip, which may be the same chip as the microarray chip or a different chip, and where
standard spot 1 contains only the donor fluorophore and none of the acceptor fluorophore used in the two-color fluorescence measurement system,
standard spot 2 contains only the acceptor fluorophore and none of the donor fluorophore used in the two-color fluorescence measurement system,
where the acceptor fluorophore in standard spot 2 is present in the same molar amount as the donor fluorophore in standard sample 1, and
standard spot 3 contains the donor and acceptor fluorophores together, wherein the molar quantity of the donor fluorophore in standard spot 3 equals the molar quantity of the acceptor fluorophore in standard spot 3.

32 more elements

Dependencies are inferred from the claim text.

Reliable fluorescence correction method for two-color measurement fluorescence system

Life of this patent

Abstract

Claims18

Reliable fluorescence correction method for two-color measurement fluorescence system

Life of this patent

Abstract

Claims18