Correction Method and Measurement Device For Anti-Stokes Photoluminescence Measurement

• US 20080174766A1
• Filed: 05/24/2006
• Published: 07/24/2008
• Est. Priority Date: 05/24/2005
• Status: Active Grant

• Current Assignee: Hidex Oy
• Original Assignee: Hidex Oy
• Inventors: Haaslahti, Ville, Oikari, Timo, Soukka, Tero, Aalto, Juhani
• Granted Patent: US 7,826,052 B2
• Time in Patent Office: Days
• US Class Current: 356/73
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: 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
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

This invention relates to a method to correct for error in an anti-Stokes photoluminescence measurement in a Foerster-type resonance energy-transfer assay. The method comprises the steps of exciting anti-Stokes photoluminescent molecules, ions, phosphors, chelates, or particles, i.e. donors of the assay, with one wavelength or multiple wavelengths of light wherein the wavelength or wavelengths are greater than that of an emission wavelength of acceptor molecules in the assay; measuring emission light signal at a wavelength, which is the emission wavelength of the acceptor molecules in the Foerster-type resonance energy-transfer assay, and which differs from the emission wavelength of the anti-Stokes photoluminescent donor in at least two different time windows; a first time window within the time window defined by the excitation light pulse, i.e. within the time window opening when the excitation light pulse is switched on and closing when the excitation light pulse is switched off, and a second time window to follow the first time window not at all overlapping with the time window defined by the excitation pulse; and correcting the emission light signal measured, comprising signals originating from non-radiative and radiative energy transfer, within the first time window by estimating the ratio of the signals from non-radiative and radiative energy transfer or the signal originating from radiative energy transfer using at least one emission light signal measured in the second time window. This invention also relates to the use of a device according to the method of the invention, a system for carrying out the method and a software product for the system.

Claims

1. A method to correct for error in an anti-Stokes photoluminescence measurement in a Foerster-type resonance energy-transfer assay, comprising the following steps

a) exciting anti-Stokes photoluminescent molecules, ions, phosphors, chelates, or particles, i.e. donors of said assay, with one wavelength or multiple wavelengths of light wherein said wavelength or wavelengths are greater than that of an emission wavelength of acceptor molecules in said assay,

b) measuring emission light signal at a wavelength, which is said emission wavelength of said acceptor molecules in said Foerster-type resonance energy-transfer assay, and which differs from the emission wavelength of said anti-Stokes photoluminescent donor, in at least two different time windows,

i) a first time window within the time window defined by the excitation light pulse, i.e. within the time window opening when the excitation light pulse is switched on and closed when the excitation light pulse is switched off, and

ii) a second time window to follow the first time window not at all overlapping with said time window defined by the excitation pulse, and

c) correcting the emission light signal measured, comprising signals originating from non-radiative and radiative energy transfer, within the first time window by estimating the ratio of the signals from non-radiative and radiative energy transfer or the signal originating from radiative energy transfer using at least one emission light signal measured in said second time window.

2. The method according to claim 1 characterized in that the second time window is within a third time window and said third time window opens as soon as the excitation light pulse has died out;

preferably 10 μ

s, more preferably 3 μ

s and most preferably 1 μ

s after having shut off the excitation light pulse; and

said third time window closes before two lifetimes of the emission of the acceptor resulting from non-radiative energy transfer, i.e. Foerster type resonance energy transfer from said donor to said acceptor, i.e. 2τ

₂, has passed from shutting off the excitation light pulse.

3. The method according to claim 1 characterized in that the second time window is within a fourth time window, which opens when at least two lifetimes of the emission of the acceptor resulting from non-radiative energy transfer, i.e. Foerster type resonance energy transfer from said donor to said acceptor, i.e. 2τ

₂, has passed from shutting off the excitation light pulse and closes before or when four lifetimes, preferably three lifetimes, and more preferably two lifetimes, of the emission of the acceptor resulting from radiative energy transfer, i.e. from reabsorbtion by the acceptor of light emitted by the donor, i.e. 4τ

₃, 3τ

₃ and 2τ

₃ correspondingly, has passed from shutting off the excitation light pulse.

4. The method according to claim 2 characterized in that said emission light is measured in at least three different time windows:

the first time window, within the time window defined by the excitation light pulse;

the third time window, which opens as soon as the excitation light pulse has died out and closes before two lifetimes of the emission of the acceptor resulting from non-radiative energy transfer, i.e. 2τ

₂, has passed from shutting off the excitation light pulse; and

the fourth time window, which opens when at least two lifetimes of the emission of the acceptor resulting from non-radiative energy transfer, i.e. 2τ

₂, has passed from shutting off the excitation light pulse and closes before or when four lifetimes, preferably three lifetimes, and more preferably two lifetimes, of the emission of the acceptor resulting from radiative energy transfer, i.e. from reabsorbtion by the acceptor of light emitted by the donor, i.e. 4τ

₃, 3τ

₃ and 2τ

₃ correspondingly, has passed from shutting off the excitation light pulse.

5. The method according to claim 1 characterized in that emission light is measured in more than three different time windows comprising multiple measurements in 1 to 100 μ

s increments within the second time window.

6. The method according to claim 5 characterized in that the multiple measurements in the 1 to 100 μ

s increments are carried out in time windows three and/or four.

7. The method according to claim 5 characterized in that the multiple measurements in the 1 to 100 μ

s increments are carried out in a fifth time window which opens as soon as the excitation light pulse has died out, preferably about 1 μ

s after having shut off the excitation light pulse; and

closes when about 50 ms, preferably 20 ms, and more preferably 5 ms has passed from shutting off the excitation light pulse.

8. Use of a device, which comprises

a) a light source, with means for turning said light source on and off either electrically or mechanically, emitting light of a wavelength that is ≧

800 nm, and

b) a detector with means for detecting light of a selected wavelength as a function of time in at least two time windows, for carrying out the method according to claim 1.

9. A system for carrying out the method according to claim 1, which system comprises

a) a light source, with means for turning said light source on and off either electrically or mechanically, emitting light of a wavelength that is >

800 nm, and

b) a detector with means for detecting light of a selected wavelength as a function of time in at least two time windows characterized in that said system further comprises a data processing unit with software for correcting an emission light signal measured within a first time window within the time window defined by an excitation light pulse, i.e. within a time window opening when the excitation light pulse is switched on and closing when the excitation light pulse is switched off, with an emission light signal measured within a second time window to follow the first time window not at all overlapping with said time window defined by the excitation pulse.

10. A software product for a system for carrying out the method according to claim 1, characterized in that the software product comprises means for correcting an emission light signal measured within a first time window within the time window defined by an excitation light pulse, i.e. within a time window opening when the excitation light pulse is switched on and closing when the excitation light pulse is switched off, with an emission light signal measured within a second time window to follow the first time window not at all overlapping with said time window defined by the excitation pulse.