Apparatus for measuring polarization of bathochromically shifted fluorescence
First Claim
1. Apparatus for the measurement of the polarization value, P, of a fluorescing material in a sample comprising of fluorescing material and background material, the background material contributing background fluorescence, where the emission spectrum of the background fluorescence is shifted relative to the emission spectrum of the fluorescence from the fluorescing material, the apparatus comprising:
- (a) an excitation source for exciting a sample comprising a fluorescing material and background material, the background contributing background fluorescence, at a selected excitation wavelength;
(b) a fixed polarizer transmitting to the sample only plane-polarized light, the polarizer being disposed between the excitation source and the sample;
(b) a fixed polarizer transmitting to the sample only plane-polarized light, the polarizer being disposed between the excitation source and the sample;
(c) orientation selection means for selectively transmitting plane-polarized light in either a first plane parallel to the plane of polarization of the exciting light or a second plane transverse to the first plane, the orientation selection means disposed in the light path of the fluorescence emitted by the sample;
(d) wavelength selection means for selecting either a primary wavelength, λ
1, or a secondary wavelength, λ
2, for subsequent fluorescence emission intensity measurements, the wavelength selection means being disposed in the path of the light emitted from the orientation selection means;
(e) measuring means for measuring the intensities of the components of the emitted fluorescence polarized in the first and second planes of the at least two wavelengths selected by the wavelength selection means; and
(f) calculation means for calculating the net polarization value, P, of the fluorescing material in the sample from the measure intensities at the at least two wavelengths.
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Abstract
An improved method and apparatus for measuring polarized fluorescence emissions compensates for background emissions without separating a fluorescing material from background material. The method involves measuring the horizontally and vertically polarized components of the fluorescence emission at a primary wavelength and at least one secondary wavelength selected on the basis of the bathochromic shift of the spectrum of the fluorescence emissions from the fluorescing material as compared to the fluorescence emissions from the background. From these measurements, a factor representing the fraction of the total intensity of fluorescence emissions due to background fluorescence is determined. From this factor, the intensities of the vertically and horizontally polarized fluorescence emissions due to background fluorescence are derived and subtracted from the measurements at the primary wavelength to obtain intensities due solely to the material being analyzed.
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Citations
37 Claims
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1. Apparatus for the measurement of the polarization value, P, of a fluorescing material in a sample comprising of fluorescing material and background material, the background material contributing background fluorescence, where the emission spectrum of the background fluorescence is shifted relative to the emission spectrum of the fluorescence from the fluorescing material, the apparatus comprising:
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(a) an excitation source for exciting a sample comprising a fluorescing material and background material, the background contributing background fluorescence, at a selected excitation wavelength; (b) a fixed polarizer transmitting to the sample only plane-polarized light, the polarizer being disposed between the excitation source and the sample; (b) a fixed polarizer transmitting to the sample only plane-polarized light, the polarizer being disposed between the excitation source and the sample; (c) orientation selection means for selectively transmitting plane-polarized light in either a first plane parallel to the plane of polarization of the exciting light or a second plane transverse to the first plane, the orientation selection means disposed in the light path of the fluorescence emitted by the sample; (d) wavelength selection means for selecting either a primary wavelength, λ
1, or a secondary wavelength, λ
2, for subsequent fluorescence emission intensity measurements, the wavelength selection means being disposed in the path of the light emitted from the orientation selection means;(e) measuring means for measuring the intensities of the components of the emitted fluorescence polarized in the first and second planes of the at least two wavelengths selected by the wavelength selection means; and (f) calculation means for calculating the net polarization value, P, of the fluorescing material in the sample from the measure intensities at the at least two wavelengths. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 30, 31)
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12. Apparatus for the measurement of a polarization value, P, of a fluorescing material in a sample comprising a fluorescing material and background material, the background material contributing background fluorescence, where the emission spectrum of the background fluorescence is shifted relative to the emission spectrum of the fluorescence form the fluorescing material, the apparatus comprising:
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(a) an excitation source for exciting a sample comprising a fluorescing material and background material, the background material contributing background fluorescence, at a selected excitation wavelength; (b) a fixed polarizer transmitting to the sample only plane-polarized light, the polarizer disposed between the excitation source and the sample; (c) orientation selection means for selectively transmitting plane-polarized light in either a first plane parallel to the plane of polarization of the exciting light or a second plane transverse to the first plane, the orientation selection means being movably disposed alternatively in the light path of a fluorescence emitted by the sample or outside of the light path; (d) wavelength selection means for selecting either a primary wavelength, λ
1, or a secondary wavelength, λ
2, for subsequent fluorescence emission intensity measurements;(e) positioning means operatively connected with the wavelength selection means such that the positioning means positions the orientation selection means in the light path only whenever the wavelength selection means select λ
1, and positions the orientation selection means outside the light path whenever the wavelength selection means select λ
2 ;(f) a first measuring means for measuring the intensity of the components of the emitted fluorescence polarized in the first and second planes λ
1 whenever the orientation selection means is disposed in the light path;(g) a second measuring means for measuring the total intensity of the fluorescence emitted from the sample at λ
2 whenever the orientation selection means is outside of the light path;(h) calculation means for calculating the net polarization value, P, of a fluorescing material in the sample from the measured intensities at the at least two wavelengths. - View Dependent Claims (13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. Apparatus for the measurement of the polarization value, P, of a fluorescing material in a sample comprising a fluorescing material and background material, the background material contributing background fluorescence, where the emission spectrum of the background fluorescence is shifted relative to the emission spectrum of the fluorescence form the fluorescing material, the apparatus comprising:
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(a) an excitation source for exciting a sample comprising a fluorescing material and background material, the background material contributing background fluorescence, at a selected excitation wavelength; (b) a fixed polarizer transmitting to the sample only vertically polarized light, the polarizer disposed between the excitation source and the sample; (c) orientation selection means for selectively transmitting plane polarized light in one of three planes; (i) a first plane parallel to the plane of polarization of the exciting light; (ii) a second plane transverse to the first plane; and (iii) a third plane oriented 54.7°
from the first plane, whereby the polarized fluorescence emission intensity measured in the third plane at a given wavelength is proportional to the total fluorescence emission intensity at that wavelength regardless of the degree of polarization of the fluorescence emitted by the samples;
the orientation means disposed in the path of the fluorescence emitted by the sample;(d) wavelength selection means for selecting either a primary wavelength, λ
1, or a secondary wavelength, λ
2, for subsequent fluorescence emission intensity measurements, the wavelength selection means operatively connected with the orientation selection means so that λ
1 is selected whenever the orientation selection means transmits light in either the first plane or the second plane, and so that λ
2 is selected whenever the orientation selection means transmits light in the third plane;(e) measuring means for measuring the intensity of the components of the emitted fluorescence polarized in the first plane, the second plane, and the third plane at the at least two wavelengths selected by the wavelength selection means; and (f) calculation means for determining It0t2, the total fluorescence emission intensity at λ
2, IM2, the polarized fluorescence emission intensity at an angle of 54.7°
to the lane of polarization, and calculating the polarization value, P, of the fluorescing material in the sample from the measured fluorescence emission intensities at the at lest two wavelengths. - View Dependent Claims (23, 24, 25, 26, 27, 28, 29, 32, 33)
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34. Apparatus for the measurement of polarization value, P, of a fluorescing material in a sample comprising a fluorescing material and background material, the background material contributing background fluorescence, where the emission spectrum of the background fluorescence is shifted relative to the emission spectrum of the fluorescence form the fluorescing material, the apparatus comprising:
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(a) an excitation source for exciting a sample comprising a fluorescing material and background material, the background material contributing background fluorescence, at a selected excitation wavelength; (b) a rotating polarizer disposed between the excitation source and the sample; (c) a first fixed orientation selection means for selectively transmitting plane-polarized light only in a first plane, the first fixed orientation selection means being disposed in the light path of the fluorescence emitted by the sample; (d) a second fixed orientation selection means for selectively transmitting plane-polarized light only in a second plane transverse to the first plane, the second fixed orientation selection means disposed in the light path of the fluorescence emitted by the sample; (e) a first wavelength selection means for selecting only a primary wavelength, λ
1, for subsequent fluorescence emission intensity measurements, the first wavelength selection means being disposed in the path of the light emitted from the first fixed orientation selection means;(f) a second wavelength selection means for selecting only a secondary wavelength, λ
2, for subsequent fluorescence emission intensity measurements, the second wavelength selection means being disposed in the path of the light emitted from the second fixed orientation selection means;(g) measuring means for measuring the intensities of the components of the emitted fluorescence polarized in the first and second planes at the at least two wavelengths selected by the first and second wavelength selection means; and (h) calculation means for calculating the net polarization value, P, of the fluorescing material in the sample from the measured intensities at the at least two wavelengths. - View Dependent Claims (35, 36, 37)
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Specification