Method and apparatus for optical spectroscopic detection of cell and tissue death

• US 20050119548A1
• Filed: 09/30/2004
• Published: 06/02/2005
• Est. Priority Date: 07/05/2002
• Status: Abandoned Application

• Current Assignee: Cleveland Clinic Foundation, Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Mahadevan-Jansen, Anita, Lin, Wei-Chiang, Toms, Steven A., Chari, Ravi S.
• Application Number:
  US10/955,331
• Publication Number:
  US 20050119548A1
• Time in Patent Office: Days
• US Class Current: 600/407
• CPC Class Codes: A61B 5/0071 by measuring fluorescence emission
• 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 5/4244 liver

Abstract

A method for detecting death process of a cell or tissue of a living subject. In one embodiment, the method includes the steps of illuminating the cell or tissue of the living subject with a coherent light, collecting fluorescent light returned from the illuminated cell or tissue of the living subject, identifying a NAD(P)H peak of a spectrum of the collected fluorescent light with a wavelength, λ_peak, and obtaining the intensity of the NAD(P)H peak of the spectrum of the collected fluorescent light substantially corresponding to the wavelength λ_peak. These steps are repeated at sequential stages until the intensity of the NAD(P)H peak of the spectrum at a current stage is less than the intensity of the NAD(P)H peak of the spectrum at an earlier stage immediately prior to the current stage so as to detect death process of the cell of the living subject at the current stage using the intensity of the NAD(P)H peak of the spectrum.

Claims

1. A method for detecting death process of a cell of a living subject, comprising the steps of:

a. illuminating the cell of the living subject with a coherent light;

b. collecting fluorescent light returned from the illuminated cell of the living subject;

c. identifying a NAD(P)H peak of a spectrum of the collected fluorescent light with a wavelength, λ

_peak;

d. obtaining the intensity of the NAD(P)H peak of the spectrum of the collected fluorescent light substantially corresponding to the wavelength λ

_peak; and

e. repeating steps (a)-(d) at sequential stages until the intensity of the NAD(P)H peak of the spectrum at a current stage M is less than the intensity of the NAD(P)H peak of the spectrum at an earlier stage M-1, wherein the stage M-1 is immediately prior to the current stage M, M being an integer greater than 1, so as to detect death process of the cell of the living subject at the current stage M using the intensity of the NAD(P)H peak of the spectrum.

2. The method of claim 1, wherein the cell of the living subject experiences apoptosis leading to death at the current stage M when the NAD(P)H peak intensity at the current stage M is less than the NAD(P)H peak intensity at the earlier stage M-1.

3. The method of claim 2, wherein the cell of the living subject is native at a first stage and is subject to hyperthermia and/or cisplatin for various periods of time at the sequential stages.

4. The method of claim 1, wherein the coherent light has a wavelength substantially at about 337 nm.

5. The method of claim 1, wherein the wavelength λ

_peak of the NAD(P)H peak is substantially at about 460 nm.

52. The method of claim 51, wherein the optical spectra include a fluorescence spectrum and a diffused reflectance spectrum.

6. A method for detecting death process of a tissue of a living subject, wherein the tissue is an aggregation of morphologically similar cells and associated intercellular matter, comprising the steps of:

a. illuminating an area of the tissue of the living subject with a coherent light;

b. collecting fluorescent light returned from the illuminated area of the tissue of the living subject;

c. identifying a NAD(P)H peak of a spectrum of the collected fluorescent light with a wavelength, λ

_peak;

d. obtaining the intensity of the NAD(P)H peak of the spectrum of the collected fluorescent light substantially corresponding to the wavelength λ

_peak; and

e. repeating steps (a)-(d) at sequential stages until the intensity of the NAD(P)H peak of the spectrum at a current stage M is less than the intensity of the NAD(P)H peak of the spectrum at an earlier stage M-1, wherein the stage M-1 is immediately prior to the current stage M, M being an integer greater than 1, so as to detect death process of the tissue of the living subject at the current stage M using the intensity of the NAD(P)H peak of the spectrum.

7. The method of claim 6, wherein the illuminated area of the tissue of the living subject experiences apoptosis leading to death at the current stage M when the NAD(P)H peak intensity at the current stage M is less than the NAD(P)H peak intensity at the earlier stage M-1.

8. The method of claim 6, wherein the tissue of the living subject is native at a first stage and is subject to hyperthermia and/or cisplatin for various periods of time at the sequential stages.

9. The method of claim 6, wherein the coherent light has a wavelength substantially at about 337 nm.

10. The method of claim 6, wherein the wavelength λ

_peak of the NAD(P)H peak is substantially at about 460 nm.

11. An apparatus for detecting death process of at least one cell of a living subject, comprising:

a. a light source adapted for emitting coherent light with a wavelength at least in a range of between 300 nm and 400 nm;

b. a fiber optical probe coupled with the light source and adapted for delivering the coherent light to the at least one cell of the living subject proximal to a working end of the fiber optical probe;

c. a detector coupled with the fiber optical probe so as to receive from the working end of the fiber optical probe fluorescent light returned from the at least one cell of the living subject in response to illumination by the coherent light and to provide a frequency spectrum of the returned fluorescent light; and

d. a controller coupled with the detector and programmed to identify a NAD(P)H peak of a frequency spectrum of the returned fluorescent light with a wavelength, λ

_peak, and the corresponding intensity of the NAD(P)H peak of the frequency spectrum of the returned fluorescent light so as to detect death process of the illuminated at least one cell of the living subject.

12. The apparatus of claim 11, wherein the light source comprises a laser.

13. The apparatus of claim 12, wherein the laser has at least one wavelength substantially at about 337 nm.

14. The apparatus of claim 11, wherein the detector comprises a spectrometer.

15. The apparatus of claim 11, wherein the wavelength λ

_peak of the NAD(P)H peak is substantially at about 460 nm.

16. The apparatus of claim 11, wherein the controller identifies the death of at least one cell of the living subject upon a detected change in the NAD(P)H peak.

17. The apparatus of claim 11, wherein the at least one cell of the living subject is separable from a tissue of a living subject, wherein the tissue is an aggregation of morphologically similar cells and associated intercellular matter.

18. The apparatus of claim 11, wherein the at least one cell of the living subject is associated with a tissue of a living subject, wherein the tissue is an aggregation of morphologically similar cells and associated intercellular matter.

19. A method for identifying an in vitro liver tissue of a living subject, comprising the steps of:

a. acquiring a fluorescence spectrum of an in vitro liver tissue to be identified;

b. acquiring a diffused reflectance spectrum of the in vitro liver tissue;

and

c. identifying a first peak and a second peak of the fluorescence spectrum, and a spectral profile of the diffused reflectance spectrum in a predetermined wavelength region, respectively, so as to identify the in vitro liver tissue, wherein the first peak of the fluorescence spectrum includes a first peak wavelength, λ

₁, and a corresponding first peak intensity, F(λ

₁), and the second peak of the fluorescence spectrum includes a second peak wavelength, λ

₂, and a corresponding second peak intensity, F(λ

₂), and wherein the spectral profile of the diffused reflectance spectrum includes a spectral shape and intensity, and the predetermined wavelength region is from about 600 nm to about 800 nm.

20. The method of claim 19, wherein the step of acquiring the fluorescence spectrum of the in vitro liver tissue to be identified comprises the steps of:

a. illuminating an area of the in vitro liver tissue with an excitation light;

and

b. collecting fluorescent light returned from the illuminated area of the in vitro liver tissue.

21. The method of claim 20, wherein the excitation light has a wavelength between about 320 nm and about 340 nm.

22. The method of claim 21, wherein the first peak wavelength λ

₁ of the fluorescence spectrum is in a range of about 370 nm to about 400 nm, and the second peak wavelength λ

₂ of the fluorescence spectrum is in a range of about 460 nm to about 500 nm.

23. The method of claim 19, wherein the step of acquiring the diffused reflectance spectrum of the in vitro liver tissue to be identified comprises the steps of:

a. illuminating an area of the in vitro liver tissue with a white light; and

b. collecting diffused reflectance light returned from the illuminated area of the in vitro liver tissue.

24. The method of claim 19, further comprising the step of identifying the in vitro liver tissue as a normal liver tissue when

a. the first peak wavelength λ

₁ and the second peak wavelength λ

₂ of the fluorescence spectrum are substantially at about 395 nm and about 470 mn, respectively, and a ratio of the corresponding first peak intensity F(λ

₁) to the corresponding second peak intensity F(λ

₂) of the fluorescence spectrum is less than one; and

b. the intensity of the diffused reflectance spectrum is substantially unchanged over the predetermined wavelength region from about 600 nm to about 800 nm.

25. The method of claim 19, further comprising the step of identifying the in vitro liver tissue as a malignant liver tissue with a cirrhotic liver tissue when

a. the first peak wavelength λ

₁ and the second peak wavelength λ

₂ of the fluorescence spectrum are substantially at about 395 nm and about 490 nm, respectively, and a ratio of the corresponding first peak intensity F(λ

₁) to the corresponding second peak intensity F(λ

₂) of the fluorescence spectrum is less than one; and

b. the intensity of the diffused reflectance spectrum is substantially monotonically decreased over the predetermined wavelength region from about 600 nm to about 800 nm.

26. The method of claim 19, further comprising the step of identifying the in vitro liver tissue as a malignant liver tissue with a colon metastasis liver tissue when a. the first peak wavelength λ

₁ and the second peak wavelength λ

₂ of the fluorescence spectrum are substantially at about 380 nm and about 480 nm, respectively, and a ratio of the corresponding first peak intensity F(λ

₁) to the corresponding second peak intensity F(λ

₂) of the fluorescence spectrum is about one or greater than one; and

b. the intensity of the diffused reflectance spectrum is monotonically decreased over the predetermined wavelength region from about 600 nm to about 800 nm.

27. An apparatus for identifying an in vitro liver tissue of a living subject, comprising:

a. a first light source adapted for emitting an excitation light;

b. a second light source adapted for emitting a white light;

c. a fiber optical probe coupled with the first light source and the second light source so as to deliver the excitation light and the white light to an area of an in vitro liver tissue to be identified proximal to a working end of the fiber optical probe, respectively;

d. a detector coupled with the fiber optical probe so as to receive from the working end of the fiber optical probe fluorescent light returned from the area of the in vitro liver tissue of the living subject in response to illumination by the excitation light and diffused reflectance light returned from the area of the in vitro liver tissue of the living subject in response to illumination by the white light, and to provide frequency spectra of the returned fluorescent light and the returned diffused reflectance light, respectively; and

e. a controller coupled with the detector and programmed to determine a first peak and a second peak of the frequency spectrum of the returned fluorescent light, and a spectral profile of the frequency spectrum of the returned diffused reflectance light in a predetermined wavelength region, respectively, so as to identify the in vitro liver tissue, wherein the first peak of the frequency spectrum of the returned fluorescent light includes a first peak wavelength, λ

₁, and a corresponding first peak intensity, F(λ

₁), and the second peak of the frequency spectrum of the returned fluorescent light includes a second peak wavelength, λ

₂, and a corresponding second peak intensity, F(λ

₂), and wherein the spectral profile of the frequency spectrum of the returned diffused reflectance light includes a spectral shape and intensity, and the predetermined wavelength region is from about 600 nm to about 800 nm.

28. The apparatus of claim 27, wherein the first light source comprises a laser adapted for emitting an excitation light with a wavelength between about 320 nm and about 340 nm.

29. The apparatus of claim 27, wherein the first peak wavelength λ

₁ of the frequency spectrum of the returned fluorescent light is in a range of about 370 nm to about 400 nm, and the second peak wavelength λ

₂ of t the frequency spectrum of the returned fluorescent light is in a range of about 460 nm to about 500 nm.

30. The apparatus of claim 27, wherein the second light source comprises a halogen light source.

31. The apparatus of claim 27, wherein the detector comprises a spectrometer.

32. The apparatus of claim 27, wherein the controller is associated with a computer.

33. A method for detecting a malignant liver tissue of in vivo liver tissues, wherein the in vivo liver tissues have at least a first area and a second area, at least one of the first area and the second area containing a malignant liver tissue, comprising the steps of:

a. acquiring a fluorescence spectrum of the in vivo liver tissues from the first area;

b. acquiring a diff-used reflectance spectrum of the in vivo liver tissues from the first area;

c. identifying a blood absorption signature in the fluorescence spectrum at a first wavelength about 540 nm, and at a second wavelength about 580 nm, and a spectral profile of the diffused reflectance spectrum in a predetermined wavelength region, respectively, wherein the blood absorption signature in the fluorescence spectrum is corresponding to a spectral valley, and wherein the spectral profile of the diffused reflectance spectrum includes a spectral shape and intensity, and the predetermined wavelength region is from about 600 nm to about 700 mn;

d. repeating steps (a)-(c) in the second area of the in vivo liver tissues;

and

e. identifying the in vivo liver tissue as a malignant liver tissue in one of the first area and the second area, wherein in the area no blood absorption signature is identified in the fluorescence spectrum at about 540 nm and about 580 nm, respectively, and the intensity of the diffused reflectance spectrum is substantially monotonically decreased over the predetermined wavelength region from about 600 nm to about 700 nm.

34. The method of claim 33, further comprising the step of identifying the in vivo liver tissue as a normal liver tissue in one of the first area and the second area, wherein in the area the blood absorption signature is identified in the fluorescence spectrum at about 540 nm and about 580 nm, respectively, and the intensity of the diffused reflectance spectrum is substantially unchanged over the predetermined wavelength region from about 600 nm to about 700 nm.

35. The method of claim 33, wherein the malignant liver tissue comprises a primary liver tumor when the intensity of the fluorescence spectrum is at least three times larger than the intensity of the fluorescence spectrum from a normal liver tissue over a wavelength range of about 400 nm to about 600 nm.

36. The method of claim 35, wherein the malignant liver tissue comprises a secondary liver tumor when the fluorescence spectrum has a first peak at a first peak wavelength about 400 nm and a second peak at a second peak wavelength about 480, respectively, and a ratio of a corresponding first peak intensity to a corresponding second peak intensity to be substantially about one.

37. An apparatus for detecting a malignant liver tissue of in vivo liver tissues, wherein the in vivo liver tissues have at least a first area and a second area, at least one of the first area and the second area containing a malignant liver tissue, comprising:

a. a first light source adapted for emitting an excitation light;

b. a second light source adapted for emitting a white light;

c. a fiber optical probe coupled with the first light source and the second light source so as to deliver the coherent light and the white light to an area of an in vivo liver tissues to be identified proximal to a working end of the fiber optical probe, respectively;

d. a detector coupled with the fiber optical probe so as to receive from the working end of the fiber optical probe fluorescent light returned from the area of the in vivo liver tissues of the living subject in response to illumination by the excitation light and diffused reflectance light returned from the area of the in vivo liver tissues of the living subject in response to illumination by the white light, and to provide frequency spectra of the returned fluorescent light and the returned diffused reflectance light, respectively; and

e. a controller coupled with the detector and programmed to determine a blood absorption signature in the frequency spectrum of the returned fluorescent light at a first wavelength about 540 nm, and at a second wavelength about 580 nm, and a spectral profile of the frequency spectrum of the returned diffused reflectance light in a predetermined wavelength region, respectively, wherein the blood absorption signature in the frequency spectrum of the returned fluorescent light is corresponding to a spectral valley, and wherein the spectral profile of the frequency spectrum of the returned diffused reflectance light includes a spectral shape and intensity, and the predetermined wavelength region is from about 600 nm to about 700 nm.

38. The apparatus of claim 37, wherein the first light source comprises a laser adapted for emitting an excitation light with a wavelength between about 320 nm and about 340 nm.

39. The apparatus of claim 37, wherein the second light source comprises a halogen light source.

40. The apparatus of claim 37, wherein the detector comprises a spectrometer.

41. The apparatus of claim 37, wherein the controller is associated with a computer.

42. A method for identifying an in vitro tissue of an organ of a living subject, comprising the steps of:

a. acquiring a fluorescence spectrum of an in vitro tissue to be identified;

b. acquiring a diffused reflectance spectrum of the in vitro tissue; and

c. identifying a first peak and a second peak of the fluorescence spectrnm, and a spectral profile of the diffused reflectance spectrum in a predetermined wavelength region, respectively, so as to identify the in vitro tissue, wherein the first peak of the fluorescence spectrum includes a first peak wavelength, λ

₁, and a corresponding first peak intensity, F(λ

₁), and the second peak of the fluorescence spectrum includes a second peak wavelength, λ

₂, and a corresponding second peak intensity, F(λ

₂), and wherein the spectral profile of the diffused reflectance spectrum includes a spectral shape and intensity.

43. The method of claim 42, wherein the step of acquiring the fluorescence spectrum of the in vitro tissue to be identified comprises the steps of:

a. illuminating an area of the in vitro tissue with an excitation light; and

b. collecting fluorescent light returned from the illuminated area of the in vitro tissue.

44. The method of claim 42, wherein the step of acquiring the diffused reflectance spectrum of the in vitro tissue to be identified comprises the steps of:

a. illuminating an area of the in vitro tissue with a white light; and

b. collecting diffused reflectance light returned from the illuminated area of the in vitro tissue.

45. A method of optimal placement of a radio frequency probe for a radio frequency ablation of a liver tumor in liver tissues of a living subject, wherein the radio frequency probe has a plurality of electrodes, each electrode adapted for transmitting a radio frequency energy applied to an area of the liver tissues in which a working end of the electrode is located, and a plurality of optical fibers adapted such that when the radio frequency probe is placed into the liver tissues, each optical fiber is adapted for an optical spectrum measurement in an area of the liver tissues in which a working end of the optical fiber is located, comprising the steps of:

a. placing the radio frequency probe into the liver tissues at an initially selected position;

b. acquiring optical spectra from each area of the liver tissues in which a working end of the plurality of optical fibers is located, respectively;

c. identifying a type of the liver tissues in each area from the acquired optical spectra corresponding to the area, respectively;

d. adjusting the position of the radio frequency probe from the initially selected position so as to find a new position if a normal liver tissue is identified in at least one area in which a working end of the plurality of optical fibers is located;

e. repeating steps (b)-(d) until no normal liver tissue is identified in any area in which a working end of the plurality of optical fibers is located in a current position of the radio frequency probe; and

f. choosing the current position as an optimal position of the radio frequency probe for a radio frequency ablation of a liver tumor in liver tissues.

46. The method of claim 45, wherein the optical spectra include a fluorescence spectrum and a diffused reflectance spectrum.

47. An apparatus of optimal placement of a radio frequency probe for a radio frequency ablation of a liver tumor in liver tissues of a living subject, comprising:

a. a first light source adapted for emitting a coherent light;

b. a second light source adapted for emitting a white light;

c. a radio frequency probe coupled with the first light source and the second light source and placed at an initially selected position in the liver tissues, wherein the radio frequency probe have a plurality of electrodes, each electrode adapted for transmitting a radio frequency energy applied to an area of the liver tumor in which a working end of the electrode is located, and a plurality of optical fibers is adapted such that when the radio frequency probe is placed into the liver tissues, each optical fiber is adapted for an optical spectrum measurement in an area of the predetermined margin in which a working end of the optical fiber is located;

d. a detector coupled with the radio frequency probe so as to acquire optical spectra from each area of the liver tissues in which a working end of the plurality of optical fibers is located, respectively; and

e. a controller coupled with the detector and programmed to identify a liver tissue type in each area of the liver tissues from the acquired optical spectra corresponding to each area, respectively, so as to determine if the placement of the radio frequency probe in the initially selected position is optimal.

48. The apparatus of claim 47, further comprising a radio frequency energy source coupled with the radio frequency probe for providing the radio frequency energy.

49. The apparatus of claim 47, wherein the detector comprises a spectrometer.

50. The apparatus of claim 47, wherein the controller is associated with a computer.

51. A method for controlling a volume of a radio frequency ablation of a liver tumor in liver tissues of a living subject intra-operatively, comprising the steps of:

a. placing a radio frequency probe into a volumetic center of a liver tumor to be ablated, wherein the radio frequency probe has a plurality of electrodes, each electrode adapted for transmitting a radio frequency energy applied to an area of the liver tumor in which a working end of the electrode is located, and a plurality of optical fibers adapted such that when the radio frequency probe is placed into the volumetic center of the liver tumor, working ends of the plurality of optical fibers are positioned at a predetermined margin of the liver tumor, each optical fiber adapted for an optical spectrum measurement in an area of the predetermined margin in which a working end of the optical fiber is located;

b. conducting a radio frequency ablation of the liver tumor with the radio frequency probe;

c. acquiring optical spectra from each area of the predetermined margin of the liver tumor in which a working end of the plurality of optical fibers is located;

d. monitoring liver tissue coagulation in each area of the predetermined margin from the acquired optical spectra corresponding to each area;

and

e. terminating the radio frequency ablation when the liver tissue coagulation in the predetermined margin appears in all monitored areas.

53. An apparatus for monitoring a volume of a radio frequency ablation of a liver tumor in liver tissues of a living subject intra-operatively, comprising:

a. at least one light source adapted for emitting a light;

b. a radio frequency energy source adapted for providing a radio frequency energy;

c. a radio frequency probe coupled with the radio frequency energy source and the at least one light source and placed at a volumetic center of a liver tumor to be ablated, wherein the radio frequency probe has a plurality of electrodes, each electrode adapted for transmitting a radio frequency energy applied to an area of the liver tumor in which a working end of the electrode is located, and a plurality of optical fibers adapted such that when the radio frequency probe is placed into the volumetic center of the liver tumor, working ends of the plurality of optical fibers are positioned at a predetermined margin of the liver tumor, each optical fiber adapted for an optical spectrum measurement in an area of the predetermined margin in which a working end of the optical fiber is located;

d. a detector coupled with the radio frequency probe so as to acquire optical spectra from each area of the predetermined margin of the liver tumor in which a working end of the plurality of optical fibers is located; and

e. a controller coupled with the detector and programmed to intra-operatively monitor liver tissue coagulation in each area of the predetermined margin from the acquired optical spectra corresponding to each area.

54. The apparatus of claim 53, wherein the at least one light source comprises a coherent light source.

55. The apparatus of claim 53, wherein the at least one light source comprises a white light source.

56. The apparatus of claim 53, wherein the detector comprises a spectrometer.

57. The apparatus of claim 53, wherein the controller is associated with a computer.

58. A probe for ablation of a tumor in tissues of a living subject, comprising:

a. a plurality of first electrodes, each electrode adapted for transmitting a radio frequency energy to an area of the tissues where a working end of a corresponding first electrode is located; and

b. at least one second electrode adapted for acquiring an optical spectrum measurement in an area of the tissues where a working end of the second electrode is located.

59. The probe of claim 58, wherein the working ends of the plurality of first electrodes are at staggered length aligned to be implanted in the tissues.

60. The probe of claim 58, wherein the second electrode comprises a working end, and an optical fiber connected to the working end.