Evanescent wave background fluorescence/absorbance detection
First Claim
1. A method for the optical analysis of a slurry, which slurry comprises:
- (a) a continuous phase which comprises a component that fluoresces at an optically detectable wavelength, and(b) a discontinuous phase which comprises a component that also fluoresces at said optically detectable wavelength,the method comprising the steps of;
(i) optically exciting said slurry with an evanescent wave having a wavelength that excites the fluorescence of said components and simultaneously(ii) detecting the intensity of the fluorescence resulting therefrom, and then(iii) relating said detected intensity of fluorescence to the concentration of said component in said continuous phase, such that said detected intensity of fluorescence is independent of the concentration of the component in said discontinuous phase that also fluoresces.
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Abstract
It is proposed to increase the utility of intracellular fluorescence and absorbance measurements for control of fermentation and cell culture by correcting on-line for background fluorescence of the media, also to be able to measure the fluorescence or absorbance of the fluid media in the presence of suspended cells or particles. The measurements could be extended to measure the optical properties of other fluids which contain suspended particles. This invention uses the characteristics of the evanescent wave at the surface of an optic waveguide. The wave penetrates into the less dense medium only to about the depth of about 1/2 wavelength. The thickness of media swept by the evanescent wave is much less than the size of a cell, thereby essentially separating the fluorescence or absorbance of the media from that of the cells. Apparatus is disclosed for carrying out the methods taught herein, including the use of an optical fiber to generate the evanescent wave and the use of a flat plate waveguide to generate it. Apparatus that can read both bulk fluorescence and evanescent wave fluorescence employs a flat plate waveguide to generate the evanescent wave and fiber optics to create an alternating dual-beam approach to generating both sets of data.
54 Citations
13 Claims
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1. A method for the optical analysis of a slurry, which slurry comprises:
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(a) a continuous phase which comprises a component that fluoresces at an optically detectable wavelength, and (b) a discontinuous phase which comprises a component that also fluoresces at said optically detectable wavelength, the method comprising the steps of; (i) optically exciting said slurry with an evanescent wave having a wavelength that excites the fluorescence of said components and simultaneously (ii) detecting the intensity of the fluorescence resulting therefrom, and then (iii) relating said detected intensity of fluorescence to the concentration of said component in said continuous phase, such that said detected intensity of fluorescence is independent of the concentration of the component in said discontinuous phase that also fluoresces.
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2. A method for the optical analysis of a slurry, which slurry comprises:
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(a) a continuous phase which comprises a component that fluoresces at an optically detectable wavelength, and (b) a discontinuous phase which comprises a component that also fluoresces at said optically detectable wavelength, the method comprising the steps of; (i) optically exciting said slurry with an evanescent wave having a wavelength that excites the fluorescence of said components and simultaneously (ii) detecting the intensity of the fluorescence resulting therefrom, and at a different but nearby time (iii) illuminating the slurry with a non-evanescent wave and simultaneously (iv) detecting the intensity of the fluorescence or absorbance resulting therefrom, and then (v) determining the difference between the two intensity values, and (vi) relating said difference to the concentration of said component in said discontinuous phase, such that said difference is independent of the concentration of the component in said continuous phase that also fluoresces. - View Dependent Claims (3, 4, 5)
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6. Apparatus for determining the fluorescence or absorbance of the discontinuous phase of a system having a continuous phase which also fluoresces or absorbs light when excited or illuminated by light at an optically detectable wavelength emitted by the apparatus, comprising:
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(a) means for exciting the continuous phase with an evanescent wave at said wavelength, and (b) means for measuring the intensity of the fluorescence or absorbance resulting from the excitation of the continuous phase, and (c) means for illuminating the system with a nonevanescent wave at said wavelength, and (c) means for measuring the intensity of the fluorescence or absorbance resulting from the illumination of the system, and (e) means for comparing an intensity measurement generated by one of the aforesaid measurement means with an intensity measurement generated by the other of the aforesaid measurement means and for generating an indication of the difference between them, such that the value of said difference is related to the concentration of said component in said discontinuous phase, and such that said value is independent of the concentration of the component in said continuous phase that also fluoresces. - View Dependent Claims (7, 8, 9, 10)
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11. Apparatus for determining the fluorescence or absorbance of the discontinuous phase of a system having a continuous phase which also fluoresces or absorbs light when excited or illuminated by light at an optically detectable wavelength emitted by the apparatus, comprising:
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(a) a source of light at said wavelength, and a plate waveguide having a face in contact with said system, (b) means for directing light from said source to said plate waveguide at an angle sufficiently oblique to said face to create an evanescent wave in said system, such that a first state of excitation of the system is created, (c) means for directing light from said source to said plate waveguide at an angle approximately perpendicular to said face, such that a second state of excitation of the system is created, (d) chopper means for alternately directing light from said source to said directing means (b) or said directing means (c), (e) means for detecting light at a wavelength emitted by the system upon fluorescence, (f) means for directing light from said plate waveguide at an angle approximately perpendicular to said face to said detection means (e), (g) means for directing light from said plate waveguide at an angle oblique to said face to said detection means (e), (h) chopper means for alternately directing light from said directing means (f) or said directing means (g) to said detection means (e), (i) means for synchronizing chopper means (d) with chopper means (h), and (j) means for processing the signal from detection means (e) to compare the value obtained during the first state of excitation with that obtained during the second state of excitation and to produce output related to the difference in said values. - View Dependent Claims (12, 13)
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Specification