Raman instrument for measuring weak signals in the presence of strong background fluorescence

• US 20070078349A1
• Filed: 10/04/2005
• Published: 04/05/2007
• Est. Priority Date: 10/04/2005
• Status: Active Grant

• Current Assignee: Nu Skin International Incorporated (Nu Skin Enterprises Incorporated)
• Original Assignee: Nu Skin International Incorporated (Nu Skin Enterprises Incorporated)
• Inventors: Ferguson, Scott, Peatross, Justin, Fralick, John, Bergeson, Scott
• Granted Patent: US 7,558,619 B2
• Time in Patent Office: Days
• US Class Current: 600/476
• CPC Class Codes: A61B 2560/0223 of calibration, e.g. protocols for calibrating sensors
• CPC Class Codes: A61B 5/0059 using light, e.g. diagnosis by transillumination, diascopy, fluorescence A61B5/0093, A61B5/1455, A61B5/02416 and A61B5/0261 take precedence
• CPC Class Codes: G01J 3/02 Details
• CPC Class Codes: G01J 3/0291 Housings; Spectrometer accessories; Spatial arrangement of elements, e.g. folded path arrangements
• CPC Class Codes: G01J 3/44 Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
• CPC Class Codes: G01N 2021/6419 Excitation at two or more wavelengths
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 21/274 Calibration, base line adjustment, drift correction
• CPC Class Codes: G01N 21/65 Raman scattering

Abstract

A method for measuring a chemical concentration in tissue has two measurement steps. First, generating a first light and illuminating a portion of the tissue with the first light; capturing a first reflected light from the tissue; directing the first reflected light to a plurality of light sensors, each light sensor measuring light at a different wavelength, that wavelength being proximate to a wavelength of an expected Raman shift wavelength for the chemical in the tissue; and obtaining a measurement from each of the light sensors, each measurement being specific to the first reflected light through that light sensor. Second, generating a second light and illuminating a portion of the tissue with the second light; capturing a second reflected light from the tissue; directing the second reflected light to the plurality of light sensors, each light sensor measuring light at a different wavelength that wavelength being proximate to a wavelength of an expected Raman shift wavelength for the chemical in the tissue; and obtaining a measurement from each of the light sensors, each measurement being specific to the second reflected light through that light sensor. The measurements of the first reflected light and the measurements of the second reflected light are used to calculate a concentration of the chemical in the tissue.

Claims

1. A method of measuring the level of a selected molecule in a target sample, comprising:

illuminating the target with light of a first wavelength F1;

illuminating the target with light of a second wavelength F2 adjacent to the first wavelength F1;

receiving from the target emitted light resulting from each of the illuminations at wavelengths F1 and F2 and sampling the intensity of the emitted light at a range of wavelengths including a characteristic Raman emission wavelength Fc associated with the selected molecule and each of the illumination wavelengths;

developing a first set of sampled intensity values for emitted light resulting from the illumination for wavelength F1, said sample including a value at or near the characteristic emission wavelength Fc associated with the selected molecule and wavelength F1, at least one sample adjacent the characteristic wavelength Fc but sufficiently displaced below that wavelength to exclude Raman emissions resulting from the illumination for wavelength F1 and at least one sample adjacent the characteristic wavelength Fc but sufficiently displaced above that wavelength to exclude Raman emissions resulting from the illumination for wavelength F1;

developing a second set of sampled intensity values for emitted light resulting from the illumination for wavelength F2, said sample including a value at or near the characteristic emission wavelength Fc associated with the selected molecule and wavelength F2, at least one sample adjacent the characteristic wavelength Fc but sufficiently displaced below that wavelength to exclude Raman emissions resulting from the illumination for wavelength F2 and at least one sample adjacent the characteristic wavelength Fc but sufficiently displaced above that wavelength to exclude Raman emissions resulting from the illumination for wavelength F2; and

deriving from the first and second sets of sampled intensity values an interpolated intensity value for emitted light between the characteristic wavelength Fc associated with each of F1 and F2 that removes the intensity value component due to non-Raman emissions.

2. The method of claim 1 wherein the step of developing a first set of sampled intensity values for emitted light resulting from the illumination for wavelength F1 comprises developing at at least four intensity sensors a sample value resulting from the illuminations for wavelength F1 {SF10, SF11, SF12, SF13} and the step of developing a second set of sampled intensity values for emitted light resulting from the illumination for wavelength F2 comprises developing at at least four intensity sensors at least four sample values resulting from the illuminations at wavelength F2 {SF20, SF21, SF22, SF23}, where each of {SF10, SF11, SF12, SF13} and {SF20, SF21, SF22, SF23} is an ordered set of values with SF11 being a measurement at characteristic emitted wavelength Fc associated with the selected molecule and F1 and SF22 being a measurement at characteristic emitted wavelength Fc associated with the selected molecule and F2.

3. The method of claim 2 further comprising adjusting each of {SF10, SF11, SF12, SF13} and {SF20, SF21, SF22, SF23} for variations in the output of the at least four intensity sensors since a calibration of such sensors.

4. The method of claim 2 further comprising adjusting each of {SF10, SF11, SF12, SF13} and {SF20, SF21, SF22, SF23} for a dark value derived from an optically dark sample target with no Raman scattering in the light emitted from the sample.

5. The method of claim 1 wherein the step of deriving from the first and second sets of sampled intensity values an interpolated intensity value comprises, computing the ratios {SF10/SF20, SF11/SF21, SF12/SF22 SF13/SF23} and from the resulting computed ratios, extrapolating by curve fitting to find a curve that passes through the points {f0, SF10/SF20} and {f3, SF13/SF23} and the curve is equidistant from the points {f1, SF11/SF21} and {f2 SF12/SF22}, where each of f0, f1, f2 and f3 is a center wavelength for a respective first, second, third and fourth of the four sensors.

6. The method of claim 5 wherein the curve fitting uses a parabola as the curve.

7. The method of claim 5, comprising from the resulting computed ratios, graphically determining in a parabolic interpolation using a curve anchored at {f0, SF10/SF20} and {f3 SF13/SF23} a value that is at the midpoint of the wavelength range for the four sensors and that lies midway between the points {f1, SF11/SF21} and {f2 SF12/SF22}.

8. The method of claim 1, comprising from the resulting computed ratios, graphically determining a value that lies midway between the points {f1, SF11/SF21} and {f2 SF12/SF22} as measured by curve fitting with a non-linear curve anchored at {f0, SF10/SF20} and {f3, SF13/SF23}.

9. The method of claim 1 wherein the step of receiving from the target emitted light resulting from each of the illuminations at wavelength F1 and F2 and sampling the intensity of the emitted light at a range of wavelengths including a characteristic Raman emitted wavelength Fc associated with the selected molecule and each of the illumination wavelengths comprises directing the emitted light in predefined portions to at least four intensity sensors.

10. The method of claim 9 wherein directing the emitted light in predefined portions to at least four intensity sensors comprises directing the emitted light in equal portions.

11. The method of claim 1 wherein the selected molecule is a carotenoid.

12. The method of claim 1 wherein the target sample is human tissue.

13. The method of claim 1 wherein the target sample is tissue on a human hand.

14. The method of claim 1 where the step of illuminating the target with light of a second wavelength F2 adjacent to the first wavelength F1 and with an intensity in a predetermined relationship to the intensity of the light of first wavelength F1 comprises illuminating the target with light of a second wavelength F2 having an intensity substantially equal to the intensity of the light of first wavelength F1.

15. The method of claim 1 wherein F1 and F2 are separated by less than three nanometers.

16. The method of claim 1 wherein F1 and F2 are separated by less than two nanometers.

17. The method of claim 1 wherein the sampled emitted intensity values cover a wavelength range of less than 10 nanometers.

18. The method of claim 1 wherein the sampled emitted intensity values cover a wavelength range of less than 7 nanometers

19. The method of claim 1 further comprising adjusting intensity of the light of first wavelength F1 and the light of the second wavelength F2 to provide equal intensity from each light at the target when the respective light is providing illumination.

20. The method of claim 1 wherein the first and second wavelengths F1 and F2 are substantially Raman resonance wavelengths.

21. An apparatus for measuring the level of a selected molecule in a target sample, comprising:

a first light source for illuminating the target with light of a first wavelength F1;

a second light source for illuminating the target with light of a second wavelength F2 adjacent to the first wavelength F1;

means for receiving from the target emitted light resulting from each of the illuminations at wavelengths F1 and F2 and sampling the intensity of the emitted light at a range of wavelengths including a characteristic Raman emission wavelength Fc associated with the selected molecule and each of the illumination wavelengths;

a first sampler for developing a first set of sampled intensity values for emitted light resulting from the illumination for wavelength F1, said sample including a value at or near the characteristic emission wavelength Fc associated with the selected molecule and F1, at least one sample adjacent the characteristic wavelength Fc but sufficiently displaced below that wavelength to exclude Raman emissions resulting from the illumination for wavelength F1 and at least one sample adjacent the characteristic wavelength Fc but sufficiently displaced above that wavelength to exclude Raman emissions resulting from the illumination for wavelength F1;

a second sampler for developing a second set of sampled intensity values for emitted light resulting from the illumination for wavelength F2, said sample including a value at or near the characteristic emission wavelength Fc associated with the selected molecule and F2, at least one sample adjacent the characteristic wavelength Fc but sufficiently displaced below that wavelength to exclude Raman emissions resulting from the illumination for wavelength F2 and at least one sample adjacent the characteristic wavelength Fc but sufficiently displaced above that wavelength to exclude Raman emissions resulting from the illumination for wavelength F2; and

logic for deriving from the first and second sets of sampled intensity values an interpolated intensity value for emitted light between the characteristic wavelength Fc associated with each of F1 and F2 that removes the intensity value component due to non-Raman emissions.

22. The apparatus of claim 21 wherein the sampler for developing a first and second set of sampled intensity values for emitted light resulting from the illumination for wavelength F1 and the wavelength F2, respectively, comprises at least four intensity sensors with logic to sense four samples value resulting from the illumination for wavelength F1 {SF10, SF11, SF12, SF13} and logic to sense four sample values resulting from the illumination at wavelength F2 {SF20, SF21, SF22, SF23}, where each of {SF10, SF11, SF12, SF13} and {SF20, SF21, SF22, SF23} is an ordered set of values with SF11 being a measurement at characteristic emitted wavelength Fc associated with the selected molecule and F1, and SF22 being a measurement at characteristic emitted wavelength Fc associated with the selected molecule and F2.

23. The apparatus of claim 22 further comprising means for adjusting each of {SF10, SF11, SF12, SF13} and {SF20, SF21, SF22, SF23} for variations in the output of the at least four intensity sensors since a calibration of such sensors.

24. The apparatus of claim 22 further comprising means for adjusting each of {SF10, SF11, SF12, SF13} and {SF20, SF21, SF22, SF23} for a dark value derived from an optically dark sample target with no Raman scattering in the light emitted from the sample.

25. The apparatus of claim 21 wherein the logic for deriving from the first and second sets of sampled intensity values an interpolated intensity value comprises, logic for computing the ratios {SF10/SF20, SF11/SF21, SF12/SF22 SF13/SF23} and from the resulting computed ratios, extrapolating by curve fitting to find a curve that passes through the points {f0, SF10/SF20} and {f3, SF13/SF23} and the curve is equidistant from the points {f1, SF11/SF21} and {f2 SF12/SF22}, where each of f0, f1, f2 and f3 is a center wavelength for a respective first, second, third and fourth of the four sensors.

26. The apparatus of claim 25 wherein the logic for curve fitting uses a parabola as the curve.

27. The apparatus of claim 25, comprising logic using the resulting computed ratios to graphically determine in a parabolic interpolation based on a curve anchored at {f0, SF10/SF20} and {f3, SF13/SF23} a value that is at the midpoint of the wavelength range for the four sensors and that lies midway between the points {f1, SF11/SF21] and {f2, SF12/SF22}.

28. The apparatus of claim 21, comprising logic using the resulting computed ratios to graphically determine a value that lies midway between the points {f1, SF11/SF21} and {f2, SF12/SF22} as measured by curve fitting with a non-linear curve anchored at {f0, SF10/SF20} and {f3, SF13/SF23}.

29. The apparatus of claim 21 wherein the means for receiving from the target emitted light resulting from each of the illuminations at wavelength F1 and F2 and sampling the intensity of the emitted light at a range of wavelengths including a characteristic Raman emitted wavelength Fc associated with the selected molecule and each of the illumination wavelengths comprises means for directing the emitted light in predefined portions to at least four intensity sensors.

30. The apparatus of claim 29 wherein the means for directing the emitted light in predefined portions to at least four intensity sensors comprises means for directing the emitted light in equal portions.

31. A method for measuring a chemical concentration in tissue comprising:

generating a first light and illuminating a portion of the tissue with the first light;

capturing a first reflected light from the tissue;

directing the first reflected light to a plurality of light sensors, each light sensor measuring light at a different wavelength, that wavelength being proximate to a wavelength of an expected Raman shift wavelength for the chemical in the tissue;

obtaining a measurement from each of the light sensors, each measurement being specific to the first reflected light through that light sensor;

generating a second light and illuminating a portion of the tissue with the second light;

capturing a second reflected light from the tissue;

directing the second reflected light to the plurality of light sensors, each light source measuring light at a different wavelength that wavelength being proximate to a wavelength of an expected Raman shift wavelength for the chemical in the tissue;

obtaining a measurement from each of the light sources, each measurement being specific to the second reflected light through that light source; and

using the measurements of the first reflected light and the measurements of the second reflected light to calculate a concentration of the chemical in the tissue.

32. The method of claim 31, wherein the first and second lights are generated by light emitting diodes.

33. The method of claim 31, wherein the first and second lights are passed through first and second filters respectively.

34. The method of claim 31, wherein the first filter passes only light at 471.3 nanometers and the second filter passes only light at 473 nanometers.

35. The method of claim 34, wherein the first and second reflected lights are each divided into four equal portions and wherein the equal portions are directed to four light sensors.

36. The method of claim 35, wherein the first light sensor measures light at 505.8 nanometers, the second light sensor measures light at 507.8 nanometers, the third light sensor measures light at 509.8 nanometers, and the fourth light sensor measures light at 511.8 nanometers.

37. The method of claim 31, wherein the first and second reflected lights are directed to four light sensors.

38. The method of claim 37, wherein each of the first and second reflected lights are equally divided with an equal portion of each light being directed to each light sensor.

39. The method of claim 31, wherein using the measurements of the first reflected light and the measurements of the second reflected light to calculate a concentration of the chemical in the tissue comprises:

dividing the measurement from each of the light sensors specific to the first reflected light through that light sensor by the measurement from the respective light sensor specific to the second reflected light through that light sensor to obtain a Raman ratio; and

multiplying the Raman ratio by an average level of fluorescence from the tissue.

40. The method of claim 39, wherein the average level of fluorescence is approximated by an average PMT voltage measured when the portion of tissue is illuminated with the second light.

41. The method of claim 39, wherein the average intensity level of fluorescence from the tissue is at least fifty times greater that than the intensity of Raman shift emissions from the tissue.

42. The method of claim 31, further comprising gathering normalization data and using the normalization data with the measurements of the first reflected light and the measurements of the second reflected light to calculate a concentration of the chemical in the tissue.

43. The method of claim 31, wherein the light sensors are photomultiplier tubes.