Method and system for examining biological materials using low power CW excitation raman spectroscopy

• US 6,151,522 A
• Filed: 03/16/1999
• Issued: 11/21/2000
• Est. Priority Date: 03/16/1998
• Status: Expired due to Fees

• Current Assignee: Avery Dennison Corporation, The Research Foundation of State University of New York
• Original Assignee: The Research Foundation of State University of New York
• Inventors: Alfano, Robert R., Wang, Wubao
• Primary Examiner(s): PAIK, SANG YEOP
• Application Number:
  US09/270,383
• Time in Patent Office: 616 Days
• Field of Search: 600/473, 600/475, 600/476, 600/477, 600/310, 600/316, 600/318, 600/319, 356/39, 356/40, 356/41, 356/42, 356/300, 356/301, 356/303
• US Class Current: 600/473
• CPC Class Codes: A61B 5/0075 by spectroscopy, i.e. measuring spectra, e.g. Raman spectroscopy, infrared absorption spectroscopy A61B5/0071 takes precedence
• CPC Class Codes: A61B 5/0084 for introduction into the body, e.g. by catheters A61B5/1459 takes precedence
• CPC Class Codes: A61B 5/0086 using infrared radiation
• CPC Class Codes: A61B 5/4312 Breast evaluation or disorder diagnosis A61B5/0091 takes precedence
• CPC Class Codes: G01J 3/44 Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
• CPC Class Codes: G01N 21/65 Raman scattering

Abstract

A method and system for examining biological materials using low-power cw excitation Raman spectroscopy. In accordance with the teachings of the invention, a low-power continuous wave (cw) pump laser beam and a low-power cw Stokes (or anti-Stokes) probe laser beam simultaneously illuminate a biological material and traverse the biological material in collinearity. The pump beam, whose frequency is varied, is used to induce Raman emission from the biological material. The intensity of the probe beam, whose frequency is kept constant, is monitored as it leaves the biological material. When the difference between the pump and probe excitation frequencies is equal to a Raman vibrational mode frequency of the biological material, the weak probe signal becomes amplified by one or more orders of magnitude (typically up to about 10⁴ -10⁶) due to the Raman emission from the pump beam. In this manner, by monitoring the intensity of the probe beam emitted from the biological material as the pump beam is varied in frequency, one can obtain an excitation Raman spectrum for the biological material tested. The present invention may be applied to in the in vivo and/or in vitro diagnosis of diabetes, heart disease, hepatitis, cancers and other diseases by measuring the characteristic excitation Raman lines of blood glucose, cholesterol, serum glutamic oxalacetic transaminase (SGOT)/serum glutamic pyruvic tansaminase (SGPT), tissues and other corresponding Raman-active body constituents, respectively. For example, it may also be used to diagnose diseases associated with the concentration of Raman-active constituents in urine, lymph and saliva It may be used to identify cancer in the breast, cervix, uterus, ovaries and the like by measuring the fingerprint excitation Raman spectra of these tissues. It may also be used to reveal the growing of tumors or cancers by measuring the levels of nitric oxide in tissue.

Claims

1. A method of examining an object, said method comprising the steps of:

(a) irradiating the object with a first pump beam at a first wavelength, whereby the object produces a spontaneous Raman emission at a second wavelength;

(b) substantially simultaneously with step (a), irradiating the object with a first probe beam at a third wavelength, whereby the intensity of said first probe beam emitted from the object at said third wavelength increases if said third wavelength is equal to said second wavelength;

(c) detecting the intensity of said first probe beam emitted from the object at said third wavelength following steps (a) and (b), whereby an increase in intensity of said first probe beam at said third wavelength indicates the presence of a Raman mode for the object at said first wavelength;

(d) irradiating the object with a second pump beam at a fourth wavelength, whereby the object produces a spontaneous Raman emission at a fifth wavelength, said fourth wavelength not being equal to said first wavelength;

(e) substantially simultaneously with step (d), irradiating the object with said first probe beam at said third wavelength, whereby the intensity of said first probe beam emitted from the object at said third wavelength increases if said third wavelength is equal to said fifth wavelength; and

(f) detecting the intensity of said first probe beam emitted from the object at said third wavelength following steps (d) and (e), whereby an increase in intensity of said first probe beam at said third wavelength indicates the presence of a Raman mode for the object at said fourth wavelength.

2. The method as claimed in claim 1 further comprising the steps of:

(g) irradiating the object with a pump beam at a wavelength other than said first wavelength and said fourth wavelength;

(h) substantially simultaneously with step (g), irradiating the object with said first probe beam at said third wavelength;

(i) detecting the intensity of said first probe beam emitted from the object at said third wavelength following steps (g) and (h);

(j) repeating steps (g) through (i) until a spectrum of different pump beam wavelengths have been used; and

(k) generating an excitation Raman spectrum for the object using the intensities detected in steps (c), (f) and (i).

3. The method as claimed in claim 2 wherein the object is a biological material.

4. The method as claimed in claim 2 wherein the object is a blood sample.

5. The method as claimed in claim 4 further comprising the step of comparing the detected intensities at one or more of the Raman modes for blood glucose to appropriate standards so that the concentration of blood glucose in the blood sample can be determined.

6. The method as claimed in claim 4 further comprising the step of comparing the detected intensities at one or more of the Raman modes for serum cholesterol to appropriate standards so that the concentration of serum cholesterol in the blood sample can be determined.

7. The method as claimed in claim 4 further comprising the step of comparing the detected intensities at one or more of the Raman modes for serum glutamic oxalacetic transaminase and serum glutamic pyruvic transaminase to appropriate standards so that the concentrations of serum glutamic oxalacetic transaminase and serum glutamic pyruvic transaminase in the blood sample can be determined.

8. The method as claimed in claim 4 further comprising the step of comparing the detected intensities at one or more of the Raman modes for blood urea nitrogen to appropriate standards so that the concentration of blood urea nitrogen in the blood sample can be determined.

9. The method as claimed in claim 4 further comprising the step of comparing the detected intensities at one or more of the Raman modes for creatinine to appropriate standards so that the concentration of creatinine in the blood sample can be determined.

10. The method as claimed in claim 4 further comprising the step of comparing the detected intensities at one or more of the Raman modes for acid phosphatase to appropriate standards so that the concentration of acid phosphatase in the blood sample can be determined.

11. The method as claimed in claim 3 wherein the biological material is a tissue sample, said method further comprising the step of comparing said excitation Raman spectrum to appropriate standards obtained for cancerous and noncancerous tissues of the same type.

12. The method as claimed in claim 11 wherein the tissue sample is a human breast tissue sample.

13. The method as claimed in claim 11 wherein the tissue sample is a human cervical tissue sample.

14. The method as claimed in claim 11 wherein the tissue sample is a human uterine tissue sample.

15. The method as claimed in claim 11 wherein the tissue sample is a human ovarian tissue sample.

16. The method as claimed in claim 3 wherein the biological material is a urine, lymph, saliva, semen or tear sample.

17. The method as claimed in claim 3 wherein the biological material is a human aortic tissue sample, said method further comprising the step of comparing said excitation Raman spectrum to appropriate standards obtained for normal human aortic tissue, calcified human plaque tissue and fibrous human plaque tissue.

18. The method as claimed in claim 1 wherein the object examined is part of a living being and wherein said method is performed in vivo.

19. The method as claimed in claim 1 further comprising the step of comparing the intensities detected in steps (c) and (f) to the intensity of said first probe beam emitted from the object at said third wavelength in the absence of a pump beam.

20. The method as claimed in claim 1 wherein one of said first and said fourth wavelengths is a known Raman mode for the object being examined and wherein the other of said first and said fourth wavelengths is known not to be a Raman mode for the object being examined.

21. A system for examining an object, said system comprising:

(a) means for producing a first low-power pump beam at a first wavelength;

(b) means for producing a second low-power pump beam at a second wavelength, said second wavelength differing from said first wavelength;

(c) means for producing a first low-power probe beam at a third wavelength, said third wavelength differing from each of said first and second wavelengths;

(d) optics for combining said first low-power probe beam and said first low-power pump beam into a first combined beam focused on the object and for combining said first low-power probe beam and said second low-power pump beam into a second combined beam focused on the object; and

(e) means for detecting the intensity of light emitted from the object at said third wavelength.

22. The method as claimed in claim 21 further comprising a chopper for modulating said first and second low-power pump beams prior to their combination with said first low-power probe beam and further comprising a lock-in amplifier for demodulating the light emitted from the object at said third wavelength and detected by said detecting means.