US 2003/0117705 A1 · Fluorescence polarization assay system and method

Abstract

A sample having at least one probe is illuminated with at least one beam of excitation light that is linearly polarized along a first axis, thereby effecting fluorescence emission in at least one spectral band. The intensity of a first component of fluorescence emission that is polarized along the first axis, as well as the intensity of a second component of fluorescence emission that is polarized along an orthogonal second axis, is measured for each of said at least one spectral band. These measurements are represented as a measurement vector M. Since there are various fluorescence sources besides the at least one probe which emit some limited amount of light in the characteristic band of the at least one probe, spectral cross-talk results between the at least one probe and the other fluorescence sources. The measurement vector is therefore corrected using an instrument response matrix A, which is generated by measuring the flux output of control samples which each have only a single one of the other fluorescence sources. A flux vector S is calculated according to S=A−1M, and the fluorescence polarization FP of the at least one probe is calculated from the S values.

Claims12

Dependent claims

A method for measuring the fluorescence polarization of at least one probe in a single sample, which method compensates for spectral cross-talk at least between the at least one probe and at least one fluorescence source, said method comprising the steps of:

illuminating the sample having the at least one probe with at least one linearly polarized beam of excitation light, thereby effecting fluorescence emission in at least one emission spectral band, wherein the at least one linearly polarized beam of excitation light has at least one excitation spectral band;
measuring the intensity of a first component of fluorescence emission that is linearly polarized along a first axis, in the at least one emission spectral band;
measuring the intensity of a second component of fluorescence emission that is linearly polarized along a second axis that is orthogonal to the first axis, in the at least one emission spectral band;
illuminating each of a plurality of control samples which each have only a respective one of the at least one fluorescence source with the at least one linearly polarized beam of excitation light, thereby effecting fluorescence emission in the at least one emission spectral band from each of said plurality of control samples;
measuring the intensities of said first and second components of fluorescence emission in each of the at least one spectral band for each of said control samples which each have only a respective one of the at least one fluorescence source;
and
compensating for spectral cross-talk at least between the at least one probe and the at least one fluorescence source by determining the relative contributions from each of the at least one fluorescence source to the measured intensities of said first and second components of fluorescence emission at the at least one emission spectral band, and calculating the fluorescence polarization of each of the at least one probe based on said relative contributions.

4 more elements

illuminating the sample having the at least one probe with at least one linearly polarized beam of excitation light, thereby effecting fluorescence emission in at least one emission spectral band, wherein the at least one linearly polarized beam of excitation light has at least one excitation spectral band;
measuring the intensity of a first component of fluorescence emission that is linearly polarized along a first axis, in the at least one emission spectral band;
measuring the intensity of a second component of fluorescence emission that is linearly polarized along a second axis that is orthogonal to the first axis, in the at least one emission spectral band;
and
compensating for spectral cross-talk at least between the at least one probe and the at least one fluorescence source by determining the relative contributions from each of the at least one fluorescence source to the measured intensities of said first and second components of fluorescence emission at the at least one emission spectral band, and calculating the fluorescence polarization of each of the at least one probe based on said relative contributions.

2 more elements

A method for measuring at least one of fluorescence polarization and fluorescence anisotropy of a plurality of probes in a measurement sample, which method compensates for spectral cross-talk among said plurality of probes, said method comprising the steps of:

obtaining values for an instrumental response matrix A for an excitation and emission polarization state pair, using a plurality of control samples, wherein each control sample contains only one probe and each value in instrumental response matrix A corresponds to a spectral band of a plurality of spectral bands and a probe of the plurality of probes;
obtaining values for measurement vector M for the excitation and emission polarization state pair, using the measurement sample, wherein each value in measurement vector M corresponds to a spectral band of the plurality of spectral bands;
calculating flux vector S for the excitation and emission polarization state pair, wherein each value in flux vector S corresponds to a probe of the plurality of probes, using the following equation:
S=A−1M;
and
using the calculated S values to calculate at least one of fluorescence polarization and fluorescence anisotropy of the plurality of probes in the measurement sample.

3 more elements

Dependencies are inferred from the claim text.

Fluorescence polarization assay system and method

Life of this patent

Fluorescence polarization assay system and method

Life of this patent

Abstract

Claims12